Oligonucleotide compositions and methods thereof

ABSTRACT

Among other things, the present disclosure relates to designed oligonucleotides, compositions, and methods thereof. In some embodiments, provided oligonucleotide compositions provide altered splicing of a transcript. In some embodiments, provided oligonucleotide compositions have low toxicity. In some embodiments, provided oligonucleotide compositions provide improved protein binding profiles. In some embodiments, provided oligonucleotide compositions have improved delivery. In some embodiments, provided oligonucleotide compositions have improved uptake. In some embodiments, the present disclosure provides methods for treatment of diseases using provided oligonucleotide compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/746,199, filed Jan. 19, 2018, which is a National Stage Entry ofInternational Application No. PCT/US2016/043598, filed Jul. 22, 2016,which claims priority to United States Provisional Application Nos.62/195,779, filed Jul. 22, 2015, 62/236,847, filed Oct. 2, 2015, and62/331,960, filed May 4, 2016, the entirety of each of which isincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 8, 2016, isnamed Sequence Listing.txt and is 29,864 bytes in size.

BACKGROUND

Oligonucleotides are useful in therapeutic, diagnostic, research andnanomaterials applications. The use of naturally occurring nucleic acids(e.g., unmodified DNA or RNA) for therapeutics can be limited, forexample, because of their instability against extra- and intracellularnucleases, toxicity, and/or their poor cell penetration anddistribution. There is a need for new and improved oligonucleotides andoligonucleotide compositions, such as, e.g., new antisense and siRNAoligonucleotides and oligonucleotide compositions.

SUMMARY

Among other things, the present disclosure encompasses the recognitionthat structural elements of oligonucleotides, such as base sequence,chemical modifications (e.g., modifications of sugar, base, and/orinternucleotidic linkages, and patterns thereof), and/or stereochemistry(e.g., stereochemistry of backbone chiral centers (chiralinternucleotidic linkages), and/or patterns thereof), can havesignificant impact on oligonucleotide properties, e.g., activities,toxicities, e.g., as may be mediated by protein binding characteristics,stability, etc. In some embodiments, the present disclosure demonstratesthat oligonucleotide compositions comprising oligonucleotides withcontrolled structural elements, e.g., controlled chemical modificationand/or controlled backbone stereochemistry patterns, provide unexpectedproperties, including but not limited to certain activities, toxicities,etc. In some embodiments, the present disclosure demonstrates thatoligonucleotide properties, e.g., activities, toxicities, etc., can bemodulated by chemical modifications (e.g., modifications of sugars,bases, internucleotidic linkages, etc.), chiral structures (e.g.,stereochemistry of chiral internucleotidic linkages and patternsthereof, etc.), and/or combination thereof.

The present disclosure recognizes challenges of providing low toxicityoligonucleotide compositions and methods thereof. In some embodiments,the present disclosure provides oligonucleotide compositions and methodswith reduced toxicity. In some embodiments, the present disclosureprovides oligonucleotide compositions and methods with reduced immuneresponses. In some embodiments, the present disclosure recognizes thatvarious toxicities induced by oligonucleotides are related to complementactivation. In some embodiments, the present disclosure providesoligonucleotide compositions and methods with reduced complementactivation. In some embodiments, the present disclosure providesoligonucleotide compositions and methods with reduced complementactivation via the alternative pathway. In some embodiments, the presentdisclosure provides oligonucleotide compositions and methods withreduced complement activation via the classical pathway. In someembodiments, the present disclosure provides oligonucleotidecompositions and methods with reduced drug-induced vascular injury. Insome embodiments, the present disclosure provides oligonucleotidecompositions and methods with reduced injection site inflammation. Insome embodiments, reduced toxicity can be evaluated through one or moreassays widely known to and practiced by a person having ordinary skillin the art, e.g., evaluation of levels of complete activation product,protein binding, etc. as described herein.

In some embodiments, the present disclosure demonstrates thatoligonucleotide properties, e.g., activities, toxicities, etc., can bemodulated through chemical modifications. In some embodiments, thepresent disclosure provides an oligonucleotide composition comprising afirst plurality of oligonucleotides which have a common base sequence,and comprise one or more modified sugar moieties, one or more naturalphosphate linkages, or combinations thereof. In some embodiments, thepresent disclosure provides an oligonucleotide composition comprising afirst plurality of oligonucleotides which have a common base sequence,comprise one or more modified internucleotidic linkages, and compriseone or more modified sugar moieties, one or more natural phosphatelinkages, or combinations thereof. In some embodiments, oligonucleotidesof a first plurality have a wing-core-wing structure. In someembodiments, each wing region independently comprises one or morenatural phosphate linkages and optionally one or more modifiedinternucleotidic linkages, and the core comprises one or more modifiedinternucleotidic linkages and optionally one or more natural phosphatelinkages. In some embodiments, each wing region independently comprisesone or more natural phosphate linkages and one or more modifiedinternucleotidic linkages, and the core comprises one or more modifiedinternucleotidic linkages and no natural phosphate linkages. In someembodiments, a wing comprises modified sugar moieties. In someembodiments, a modified internucleotidic linkage is phosphorothioate. Insome embodiments, a modified internucleotidic linkage is substitutedphosphorothioate. In some embodiments, a modified internucleotidiclinkage has the structure of formula I described in this disclosure. Insome embodiments, a modified sugar moiety is 2′-modified. In someembodiments, a 2′-modification is 2′-OR′. In some embodiments, suchprovided compositions have lower toxicity. In some embodiments, providedcompositions have lower complement activation.

In some embodiments, the present disclosure provides oligonucleotidecompositions with improved protein binding profiles, e.g., loweredharmful protein binding and/or increased beneficial protein binding. Insome embodiments, the present disclosure provides methods for improveddelivery of oligonucleotide compositions comprising providingoligonucleotide compositions with improved protein binding profile. Insome embodiments, the present disclosure demonstrates that proteinbinding by oligonucleotide compositions can be modulated throughchemical modifications, stereochemistry, or combinations thereof. Insome embodiments, protein binding by oligonucleotide compositions can bemodulated by incorporation of modified internucleotidic linkages. Insome embodiments, increased percentage of modified internucleotidiclinkages provides increased binding of oligonucleotides to certainproteins. In some embodiments, replacement of one or more modifiedinternucleotidic linkages with natural phosphate linkages providesdecreased binding to certain proteins. In some embodiments, replacementof one or more natural phosphate linkages with modified internucleotidiclinkages provides increased binding to certain proteins. In someembodiments, certain chemical modifications provide increased proteinbinding to certain proteins. In some embodiments, certain chemicalmodifications provide decreased protein binding to certain proteins. Insome embodiments, different chemical modifications of the same kindprovide different protein binding. For example, in some embodiments,2′-MOE provides decreased protein binding compared to 2′-OMe (at leastin certain contexts e.g., sequence, stereochemistry, etc.).

Among other things, the present disclosure encompasses the recognitionthat stereorandom oligonucleotide preparations contain a plurality ofdistinct chemical entities that differ from one another, e.g., in thestereochemical structure of individual backbone chiral centers withinthe oligonucleotide chain. Without control of stereochemistry ofbackbone chiral centers, stereorandom oligonucleotide preparationsprovide uncontrolled compositions comprising undetermined levels ofoligonucleotide stereoisomers. Even though these stereoisomers may havethe same base sequence and/or chemical modifications, they are differentchemical entities at least due to their different backbonestereochemistry, and they can have, as demonstrated herein, differentproperties, e.g., activities, toxicities, etc. Among other things, thepresent disclosure provides chirally controlled compositions that are orcontain particular stereoisomers of oligonucleotides of interest. Insome embodiments, a particular stereoisomer may be defined, for example,by its base sequence, its length, its pattern of backbone linkages, andits pattern of backbone chiral centers. As is understood in the art, insome embodiments, base sequence may refer to the identity and/ormodification status of nucleoside residues (e.g., of sugar and/or basecomponents, relative to standard naturally occurring nucleotides such asadenine, cytosine, guanosine, thymine, and uracil) in an oligonucleotideand/or to the hybridization character (i.e., the ability to hybridizewith particular complementary residues) of such residues. In someembodiments, the present disclosure demonstrates that propertyimprovements (e.g., improved activities, lower toxicities, etc.)achieved through inclusion and/or location of particular chiralstructures within an oligonucleotide can be comparable to, or evenbetter than those achieved through use of chemical modification, e.g.,particular backbone linkages, residue modifications, etc. (e.g., throughuse of certain types of modified phosphates [e.g., phosphorothioate,substituted phosphorothioate, etc.], sugar modifications [e.g.,2′-modifications, etc.], and/or base modifications [e.g., methylation,etc.]).

Among other things, the present disclosure demonstrates thatstereochemistry can be used to modulate toxicity of oligonucleotidecompositions. In some embodiments, the present disclosure provideschirally controlled oligonucleotide compositions that have lowertoxicity when compared to a corresponding stereorandom (or chirallyuncontrolled) oligonucleotide composition of oligonucleotides sharingthe same base sequence and chemical modifications. In some embodiments,chirally controlled oligonucleotide compositions of oligonucleotidescomprising more Rp chiral internucleotidic linkage have lower toxicity.In some embodiments, chirally controlled oligonucleotide compositions ofoligonucleotides having a single Rp chiral internucleotidic linkage haveincreased toxicity compared to other chirally controlled oligonucleotidecompositions and/or the corresponding stereorandom oligonucleotidecomposition of oligonucleotides sharing the same base sequence andchemical modifications. In some embodiments, a single Rp chiralinternucleotidic linkage is in the middle of a sequence. In someembodiments, chirally controlled oligonucleotide compositions ofoligonucleotides which comprise one or more Rp chiral internucleotidiclinkages at the 5′- and/or the 3′-end provide lower toxicity. In someembodiments, chirally controlled oligonucleotide compositions ofoligonucleotides which comprise one or more natural phosphate linkagesat the 5′- and/or the 3′-end provide lower toxicity. In someembodiments, a chiral internucleotidic linkage has the structure offormula I. In some embodiments, a chiral internucleotidic linkage is aphosphorothioate linkage. In some embodiments, a chiral internucleotidiclinkage is a substituted phosphorothioate linkage.

Among other things, the present disclosure recognizes that, in someembodiments, properties (e.g., activities, toxicities, etc.) of anoligonucleotide can be adjusted by optimizing its pattern of backbonechiral centers, optionally in combination with adjustment/optimizationof one or more other features (e.g., chemical modifications, patterns ofmodifications such as linkage pattern, nucleoside modification pattern,etc.) of the oligonucleotide. In some embodiments, the presentdisclosure recognizes and demonstrates that chemical modifications, suchas modifications of nucleosides and internucleotidic linkages, canprovide enhanced properties. In some embodiments, the present disclosuredemonstrates that combinations of chemical modifications andstereochemistry can provide unexpected, greatly improved properties(e.g., activities, toxicities, etc.). In some embodiments, chemicalcombinations, such as modifications of sugars, bases, and/orinternucleotidic linkages, are combined with stereochemistry patterns toprovide oligonucleotides and compositions thereof with surprisinglyenhanced properties including low toxicity, better protein bindingprofile, etc. In some embodiments, a provided oligonucleotidecomposition comprising a first plurality of oligonucleotides is chirallycontrolled, and oligonucleotides of the first plurality comprise acombination of 2′-modification of one or more sugar moieties, one ormore natural phosphate linkages, and one or more chiral internucleotidiclinkages. In some embodiments, a provided oligonucleotide compositioncomprising a first plurality of oligonucleotides is chirally controlled,and oligonucleotides of the first plurality comprise a combination of2′-modification of one or more sugar moieties, one or more naturalphosphate linkages, one or more chiral internucleotidic linkages,wherein the 5′- and/or the 3′-end internucleotidic linkages are chiral.In some embodiments, both the 5′- and the 3′-end internucleotidiclinkages are chiral. In some embodiments, both the 5′- and the 3′-endinternucleotidic linkages are chiral and Sp. In some embodiments, aprovided oligonucleotide composition comprising a first plurality ofoligonucleotides is chirally controlled, and oligonucleotides of thefirst plurality comprise a combination of 2′-modification of one or moresugar moieties, one or more natural phosphate linkages, one or morechiral internucleotidic linkages, and a stereochemistry pattern of(Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, wherein m>2. In someembodiments, a chiral internucleotidic linkage has the structure offormula I. In some embodiments, a chiral internucleotidic linkage is aphosphorothioate linkage. In some embodiments, a chiral internucleotidiclinkage is a substituted phosphorothioate linkage.

In some embodiments, the present disclosure provides oligonucleotidecompositions having low toxicity. In some embodiments, the presentdisclosure provides oligonucleotide compositions having improved proteinbinding profile. In some embodiments, the present disclosure providesoligonucleotide compositions having improved binding to albumin. In someembodiments, provided compositions have low toxicity and improvedbinding to certain desired proteins. In some embodiments, providedcompositions have low toxicity and improved binding to certain desiredproteins. In some embodiments, provided oligonucleotide compositions atthe same time provides the same level of, or greatly enhanced, stabilityand/or activities, e.g., better target-cleavage pattern, bettertarget-cleavage efficiency, better target specificity, etc.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and optionally one or more natural phosphatelinkages, and the core region independently comprises one or moremodified internucleotidic linkages; or

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand optionally one or more natural phosphate linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingtwo wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingtwo wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages;

the wing region to the 5′-end of the core region comprises at least onemodified internucleotidic linkage followed by a natural phosphatelinkage in the wing; and

the wing region to the 3′-end of the core region comprises at least onemodified internucleotidic linkage preceded by a natural phosphatelinkage in the wing;

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisinga wing region and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

the wing region has a length of two or more bases, and comprises one ormore modified internucleotidic linkages and one or more naturalphosphate linkages;

the wing region is to the 5′-end of the core region and comprises anatural phosphate linkage between the two nucleosides at its 3′-end, orthe wing region to the 3′-end of the core region and comprises a naturalphosphate linkage between the two nucleosides at its 5′-end; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingtwo wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages;

the wing region to the 5′-end of the core region comprises a naturalphosphate linkage between the two nucleosides at its 3′-end;

the wing region to the 3′-end of a core region comprises a naturalphosphate linkage between the two nucleosides at its 5′-end; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and optionally one or more natural phosphatelinkages, and the core region independently comprises one or moremodified internucleotidic linkages; and

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and one or more natural phosphate linkages,and the core region independently comprises one or more modifiedinternucleotidic linkages; and

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides which:

1) have a common base sequence; and

2) comprise one or more wing regions and a core region; wherein:

each wing region comprises at least one modified sugar moiety; and

each core region comprises at least one un-modified sugar moiety.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotidesdefined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that apredetermined level of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising oligonucleotides of aparticular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that atleast about 10% of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

Among other things, the present disclosure recognizes that combinationsof oligonucleotide structural elements (e.g., patterns of chemicalmodifications, backbone linkages, backbone chiral centers, and/orbackbone phosphorus modifications) can provide surprisingly improvedproperties such as low toxicity and/or desired protein binding. In someembodiments, the present disclosure provides an oligonucleotidecomposition comprising a predetermined level of oligonucleotides whichcomprise one or more wing regions and a common core region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages;

the core region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages,and the common core region has:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

A wing and core can be defined by any structural elements. In someembodiments, a wing and core is defined by nucleoside modifications,wherein a wing comprises a nucleoside modification that the core regiondoes not have. In some embodiments, oligonucleotides in providedcompositions have a wing-core structure of nucleoside modification. Insome embodiments, oligonucleotides in provided compositions have acore-wing structure of nucleoside modification. In some embodiments,oligonucleotides in provided compositions have a wing-core-wingstructure of nucleoside modification. In some embodiments, a wing andcore is defined by modifications of the sugar moieties. In someembodiments, a wing and core is defined by modifications of the basemoieties. In some embodiments, each sugar moiety in the wing region hasthe same 2′-modification which is not found in the core region. In someembodiments, each sugar moiety in the wing region has the same2′-modification which is different than any sugar modifications in thecore region. In some embodiments, each sugar moiety in the wing regionhas the same 2′-modification, and the core region has no2′-modifications. In some embodiments, when two or more wings arepresent, each sugar moiety in a wing region has the same2′-modification, yet the common 2′-modification in a first wing regioncan either be the same as or different from the common 2′-modificationin a second wing region. In some embodiments, a wing and core is definedby pattern of backbone internucleotidic linkages. In some embodiments, awing comprises a type of internucleotidic linkage, and/or a pattern ofinternucleotidic linkages, that are not found in a core. In someembodiments, a wing region comprises both a modified internucleotidiclinkage and a natural phosphate linkage. In some embodiments, theinternucleotidic linkage at the 5′-end of a wing to the 5′-end of thecore region is a modified internucleotidic linkage. In some embodiments,the internucleotidic linkage at the 3′-end of a wing to the 3′-end ofthe core region is a modified internucleotidic linkage. In someembodiments, a modified internucleotidic linkage is a chiralinternucleotidic linkage.

In some embodiments, each wing comprises at least one chiralinternucleotidic linkage and at least one natural phosphate linkage. Insome embodiments, each wing comprises at least one modified sugarmoiety. In some embodiments, each wing sugar moiety is modified. In someembodiments, a wing sugar moiety is modified by a modification that isabsent from the core region. In some embodiments, a wing region only hasmodified internucleotidic linkages at one or both of its ends. In someembodiments, a wing region only has a modified internucleotidic linkageat its 5′-end. In some embodiments, a wing region only has a modifiedinternucleotidic linkage at its 3′-end. In some embodiments, a wingregion only has modified internucleotidic linkages at its 5′- and3′-ends. In some embodiments, a wing is to the 5′-end of a core, and thewing only has a modified internucleotidic linkage at its 5′-end. In someembodiments, a wing is to the 5′-end of a core, and the wing only has amodified internucleotidic linkage at its 3′-end. In some embodiments, awing is to the 5′-end of a core, and the wing only has modifiedinternucleotidic linkages at both its 5′- and 3′-ends. In someembodiments, a wing is to the 3′-end of a core, and the wing only has amodified internucleotidic linkage at its 5′-end. In some embodiments, awing is to the 3′-end of a core, and the wing only has a modifiedinternucleotidic linkage at its 3′-end. In some embodiments, a wing isto the 3′-end of a core, and the wing only has modified internucleotidiclinkages at both its 5′- and 3′-ends.

In some embodiments, each internucleotidic linkage within a core regionis modified. In some embodiments, each internucleotidic linkage within acore region is chiral. In some embodiments, a core region comprises apattern of backbone chiral centers of (Sp)m(Rp)n, (Rp)n(Sp)m,(Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, the pattern ofbackbone chiral centers of a core region is (Sp)m(Rp)n, (Rp)n(Sp)m,(Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, a core regioncomprises a pattern of backbone chiral centers of (Rp)n(Sp)m,(Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, wherein m>2. In some embodiments,the pattern of backbone chiral centers of a core region is (Sp)m(Rp)n,(Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, wherein m>2. Amongother things, in some embodiments such patterns can provide or enhancecontrolled cleavage of a target sequence, e.g., an RNA sequence.

In some embodiments, oligonucleotides in provided compositions have acommon pattern of backbone phosphorus modifications. In someembodiments, a provided composition is an oligonucleotide compositionthat is chirally controlled in that the composition contains apredetermined level of oligonucleotides of an individual oligonucleotidetype, wherein an oligonucleotide type is defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

As noted above and understood in the art, in some embodiments, basesequence of an oligonucleotide may refer to the identity and/ormodification status of nucleoside residues (e.g., of sugar and/or basecomponents, relative to standard naturally occurring nucleotides such asadenine, cytosine, guanosine, thymine, and uracil) in theoligonucleotide and/or to the hybridization character (i.e., the abilityto hybridize with particular complementary residues) of such residues.

In some embodiments, a particular oligonucleotide type may be defined by

1A) base identity;

1B) pattern of base modification;

1C) pattern of sugar modification;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

Thus, in some embodiments, oligonucleotides of a particular type mayshare identical bases but differ in their pattern of base modificationsand/or sugar modifications. In some embodiments, oligonucleotides of aparticular type may share identical bases and pattern of basemodifications (including, e.g., absence of base modification), butdiffer in pattern of sugar modifications.

In some embodiments, oligonucleotides of a particular type arechemically identical in that they have the same base sequence (includinglength), the same pattern of chemical modifications to sugar and basemoieties, the same pattern of backbone linkages (e.g., pattern ofnatural phosphate linkages, phosphorothioate linkages, phosphorothioatetriester linkages, and combinations thereof), the same pattern ofbackbone chiral centers (e.g., pattern of stereochemistry (Rp/Sp) ofchiral internucleotidic linkages), and the same pattern of backbonephosphorus modifications (e.g., pattern of modifications on theinternucleotidic phosphorus atom, such as —S⁻, and -L-R¹ of formula I).

In some embodiments, the present disclosure provides chirally controlledoligonucleotide compositions of oligonucleotides comprising multiple(e.g., more than 5, 6, 7, 8, 9, or 10) internucleotidic linkages, andparticularly for oligonucleotides comprising multiple (e.g., more than5, 6, 7, 8, 9, or 10) chiral internucleotidic linkages. In someembodiments, in a stereorandom or racemic preparation ofoligonucleotides, at least one chiral internucleotidic linkage is formedwith less than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. Insome embodiments, for a stereoselective or chirally controlledpreparation of oligonucleotides, each chiral internucleotidic linkage isformed with greater than 90:10, 95:5, 96:4, 97:3, or 98:2diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 95:5diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 96:4diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 97:3diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 98:2diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 99:1diastereoselectivity. In some embodiments, diastereoselectivity of achiral internucleotidic linkage in an oligonucleotide may be measuredthrough a model reaction, e.g. formation of a dimer under essentiallythe same or comparable conditions wherein the dimer has the sameinternucleotidic linkage as the chiral internucleotidic linkage, the5′-nucleoside of the dimer is the same as the nucleoside to the 5′-endof the chiral internucleotidic linkage, and the 3′-nucleoside of thedimer is the same as the nucleoside to the 3′-end of the chiralinternucleotidic linkage.

Among other things, the present disclosure provides oligonucleotidecompositions and technologies for optimizing properties, e.g.,activities, toxicities, etc. In some embodiments, the present disclosureprovides methods for lowering toxicity of oligonucleotides andcompositions thereof. In some embodiments, the present disclosureprovides methods for lowering immune response associated withadministration of oligonucleotides and compositions thereof (i.e., ofadministering oligonucleotide compositions so that undesirable immuneresponses to oligonucleotides in the compositions are reduced, forexample relative to those observed with a reference composition ofnucleotides of comparable or identical nucleotide sequence). In someembodiments, the present disclosure provides methods for loweringcomplement activation associated with administration of oligonucleotidesand compositions thereof. In some embodiments, the present disclosureprovides methods for improving protein binding profile ofoligonucleotides and compositions thereof. In some embodiments, thepresent disclosure provides methods for increasing binding to certainproteins by oligonucleotides and compositions thereof. In someembodiments, the present disclosure provides methods for increasingbinding to certain proteins by oligonucleotides and compositionsthereof. In some embodiments, the present disclosure provides methodsfor enhancing delivery of oligonucleotides and compositions thereof.Among other things, the present disclosure encompasses the recognitionthat optimal delivery of oligonucleotides to their targets, in someembodiments, involves balance of oligonucleotides binding to certainproteins so that oligonucleotides can be transported to the desiredlocations, and oligonucleotide release so that oligonucleotides can beproperly released from certain proteins to perform their desiredfunctions, for example, hybridization with their targets, cleavage oftheir targets, inhibition of translation, modulation of transcriptprocessing, etc. As exemplified in this disclosure, the presentdisclosure recognizes, among other things, that improvement ofoligonucleotide properties can be achieved through chemicalmodifications and/or stereochemistry.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is chirally controlled and that ischaracterized by reduced toxicity relative to a referenceoligonucleotide composition of the same common nucleotide sequence.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition in which eacholigonucleotide in the plurality comprises one or more modified sugarmoieties and the composition is characterized by reduced toxicityrelative to a reference oligonucleotide composition of the same commonnucleotide sequence but lacking at least one of the one or more modifiedsugar moieties.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition in which eacholigonucleotide in the plurality includes one or more natural phosphatelinkages and one or more modified phosphate linkages;

wherein the oligonucleotide composition is characterized by reducedtoxicity when tested in at least one assay that is observed with anotherwise comparable reference composition whose oligonucleotides do notcomprise natural phosphate linkages.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition in which eacholigonucleotide in the plurality comprises one or more modified sugarmoieties and the composition is characterized by reduced toxicityrelative to a reference oligonucleotide composition of the same commonnucleotide sequence but lacking at least one of the one or more modifiedsugar moieties.

In some embodiments, the present disclosure provides a method comprisingsteps of administering to a subject an oligonucleotide compositioncomprising a first plurality of oligonucleotides each of which has acommon base sequence and comprises a modified sugar moiety, wherein theoligonucleotide composition is characterized by reduced toxicity whentested in at least one assay that is observed with an otherwisecomparable reference composition that comprises a reference plurality ofoligonucleotides which have the same common base sequence but have nomodified sugar moieties.

In some embodiments, the present disclosure provides a method comprisingsteps of administering to a subject an oligonucleotide compositioncomprising a first plurality of oligonucleotides each of which has acommon base sequence and comprises one or more natural phosphatelinkages and one or more modified phosphate linkages, wherein theoligonucleotide composition is characterized by reduced toxicity whentested in at least one assay that is observed with an otherwisecomparable reference composition that comprises a reference plurality ofoligonucleotides which have the same common base sequence but have nonatural phosphate linkages.

In some embodiments, the present disclosure provides a method comprisingsteps of administering a chirally controlled oligonucleotide compositionto a subject, wherein the chirally controlled oligonucleotidecomposition is characterized by reduced toxicity when tested in at leastone assay that is observed with an otherwise comparable referencecomposition that includes a different chirally controlledoligonucleotide composition, or a stereorandom oligonucleotidecomposition, comprising oligonucleotides having the same base sequence.

In some embodiments, reduced toxicity is or comprises reduced complementactivation. In some embodiments, reduced toxicity comprises reducedcomplement activation. In some embodiments, reduced toxicity is orcomprises reduced complement activation. In some embodiments, reducedtoxicity comprises reduced complement activation via the alternativepathway.

In some embodiments, oligonucleotides can elicit proinflammatoryresponses. In some embodiments, the present disclosure providescompositions and methods for reducing inflammation. In some embodiments,the present disclosure provides compositions and methods for reducingproinflammatory responses. In some embodiments, the present disclosureprovides methods for reducing injection site inflammation using providedcompositions. In some embodiments, the present disclosure providesmethods for reducing drug-induced vascular injury using providedcompositions.

In some embodiments, the present disclosure provides a method,comprising administering a composition comprising a first plurality ofoligonucleotides, which composition displays reduced injection siteinflammation as compared with a reference composition comprising aplurality of oligonucleotides, each of which also has the common basesequence but which differs structurally from the oligonucleotides of thefirst plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide comprising a first plurality ofoligonucleotides that is characterized by reduced injection siteinflammation relative to a reference oligonucleotide composition of thesame common nucleotide sequence.

In some embodiments, the present disclosure provides a method,comprising administering a composition comprising a first plurality ofoligonucleotides, which composition displays altered protein binding ascompared with a reference composition comprising a plurality ofoligonucleotides, each of which also has the common base sequence butwhich differs structurally from the oligonucleotides of the firstplurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition comprising a firstplurality of oligonucleotides that is characterized by altered proteinbinding relative to a reference oligonucleotide composition of the samecommon nucleotide sequence.

In some embodiments, the present disclosure provides a method comprisingadministering a composition comprising a first plurality ofoligonucleotides, which composition displays improved delivery ascompared with a reference composition comprising a plurality ofoligonucleotides, each of which also has the common base sequence butwhich differs structurally from the oligonucleotides of the firstplurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide comprising a first plurality ofoligonucleotides that is characterized by improved delivery relative toa reference oligonucleotide composition of the same common nucleotidesequence.

In general, properties of oligonucleotide compositions as describedherein can be assessed using any appropriate assay. Relative toxicityand/or protein binding properties for different compositions (e.g.,stereocontrolled vs non-stereocontrolled, and/or differentstereocontrolled compositions) are typically desirably determined in thesame assay, in some embodiments substantially simultaneously and in someembodiments with reference to historical results.

Those of skill in the art will be aware of and/or will readily be ableto develop appropriate assays for particular oligonucleotidecompositions. The present disclosure provides descriptions of certainparticular assays, for example that may be useful in assessing one ormore features of oligonucleotide composition behavior e.g., complementactivation, injection site inflammation, protein biding, etc.

For example, certain assays that may be useful in the assessment oftoxicity and/or protein binding properties of oligonucleotidecompositions may include any assay described and/or exemplified herein.

Definitions

Aliphatic: The term “aliphatic” or “aliphatic group”, as used herein,means a straight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic or polycyclic hydrocarbon that is completely saturated orthat contains one or more units of unsaturation, but which is notaromatic (also referred to herein as “carbocycle” “cycloaliphatic” or“cycloalkyl”), that has a single point of attachment to the rest of themolecule. In some embodiments, aliphatic groups contain 1-50 aliphaticcarbon atoms. Unless otherwise specified, aliphatic groups contain 1-10aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-6 aliphatic carbon atoms. In some embodiments, aliphatic groupscontain 1-5 aliphatic carbon atoms. In other embodiments, aliphaticgroups contain 1-4 aliphatic carbon atoms. In still other embodiments,aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet otherembodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. Insome embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”)refers to a monocyclic or bicyclic C₃-C₁₀ hydrocarbon that is completelysaturated or that contains one or more units of unsaturation, but whichis not aromatic, that has a single point of attachment to the rest ofthe molecule. In some embodiments, “cycloaliphatic” (or “carbocycle” or“cycloalkyl”) refers to a monocyclic C₃-C₆ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkylene: The term “alkylene” refers to a bivalent alkyl group. An“alkylene chain” is a polymethylene group, i.e., (CH₂)_(n), wherein n isa positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3,from 1 to 2, or from 2 to 3. A substituted alkylene chain is apolymethylene group in which one or more methylene hydrogen atoms arereplaced with a substituent. Suitable substituents include thosedescribed below for a substituted aliphatic group.

Alkenylene: The term “alkenylene” refers to a bivalent alkenyl group. Asubstituted alkenylene chain is a polymethylene group containing atleast one double bond in which one or more hydrogen atoms are replacedwith a substituent. Suitable substituents include those described belowfor a substituted aliphatic group.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, a genetically-engineered animal,and/or a clone.

Approximately: As used herein, the terms “approximately” or “about” inreference to a number are generally taken to include numbers that fallwithin a range of 5%, 10%, 15%, or 20% in either direction (greater thanor less than) of the number unless otherwise stated or otherwise evidentfrom the context (except where such number would be less than 0% orexceed 100% of a possible value). In some embodiments, use of the term“about” in reference to dosages means±5 mg/kg/day.

Aryl: The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic andbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains three to seven ring members. The term “aryl”may be used interchangeably with the term “aryl ring.” In certainembodiments of the present disclosure, “aryl” refers to an aromatic ringsystem which includes, but not limited to, phenyl, biphenyl, naphthyl,anthracyl and the like, which may bear one or more substituents. Alsoincluded within the scope of the term “aryl,” as it is used herein, is agroup in which an aromatic ring is fused to one or more nonaromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a protein or polypeptide is one that contains a continuousstretch of amino acids, or a collection of continuous stretches of aminoacids, that together are characteristic of a protein or polypeptide.Each such continuous stretch generally will contain at least two aminoacids. Furthermore, those of ordinary skill in the art will appreciatethat typically at least 5, 10, 15, 20 or more amino acids are requiredto be characteristic of a protein. In general, a characteristic portionis one that, in addition to the sequence identity specified above,shares at least one functional characteristic with the relevant intactprotein.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Characteristic structural element: The term “characteristic structuralelement” refers to a distinctive structural element (e.g., corestructure, collection of pendant moieties, sequence element, etc) thatis found in all members of a family of polypeptides, small molecules, ornucleic acids, and therefore can be used by those of ordinary skill inthe art to define members of the family.

Comparable: The term “comparable” is used herein to describe two (ormore) sets of conditions or circumstances that are sufficiently similarto one another to permit comparison of results obtained or phenomenaobserved. In some embodiments, comparable sets of conditions orcircumstances are characterized by a plurality of substantiallyidentical features and one or a small number of varied features. Thoseof ordinary skill in the art will appreciate that sets of conditions arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder the different sets of conditions or circumstances are caused by orindicative of the variation in those features that are varied.

Dosing regimen: As used herein, a “dosing regimen” or “therapeuticregimen” refers to a set of unit doses (typically more than one) thatare administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, all doses within a dosing regimen are of thesame unit dose amount. In some embodiments, different doses within adosing regimen are of different amounts. In some embodiments, a dosingregimen comprises a first dose in a first dose amount, followed by oneor more additional doses in a second dose amount different from thefirst dose amount. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount same as the first dose amount.

Equivalent agents: Those of ordinary skill in the art, reading thepresent disclosure, will appreciate that the scope of useful agents inthe context of the present disclosure is not limited to thosespecifically mentioned or exemplified herein. In particular, thoseskilled in the art will recognize that active agents typically have astructure that consists of a core and attached pendant moieties, andfurthermore will appreciate that simple variations of such core and/orpendant moieties may not significantly alter activity of the agent. Forexample, in some embodiments, substitution of one or more pendantmoieties with groups of comparable three-dimensional structure and/orchemical reactivity characteristics may generate a substituted compoundor portion equivalent to a parent reference compound or portion. In someembodiments, addition or removal of one or more pendant moieties maygenerate a substituted compound equivalent to a parent referencecompound. In some embodiments, alteration of core structure, for exampleby addition or removal of a small number of bonds (typically not morethan 5, 4, 3, 2, or 1 bonds, and often only a single bond) may generatea substituted compound equivalent to a parent reference compound. Inmany embodiments, equivalent compounds may be prepared by methodsillustrated in general reaction schemes as, for example, describedbelow, or by modifications thereof, using readily available startingmaterials, reagents and conventional or provided synthesis procedures.In these reactions, it is also possible to make use of variants, whichare in themselves known, but are not mentioned here.

Equivalent Dosage: The term “equivalent dosage” is used herein tocompare dosages of different pharmaceutically active agents that effectthe same biological result. Dosages of two different agents areconsidered to be “equivalent” to one another in accordance with thepresent disclosure if they achieve a comparable level or extent of thebiological result. In some embodiments, equivalent dosages of differentpharmaceutical agents for use in accordance with the present disclosureare determined using in vitro and/or in vivo assays as described herein.In some embodiments, one or more lysosomal activating agents for use inaccordance with the present disclosure is utilized at a dose equivalentto a dose of a reference lysosomal activating agent; in some suchembodiments, the reference lysosomal activating agent for such purposeis selected from the group consisting of small molecule allostericactivators (e.g., pyrazolpyrimidines), imminosugars (e.g., isofagomine),antioxidants (e.g., n-acetyl-cysteine), and regulators of cellulartrafficking (e.g., Rabla polypeptide).

Heteroaliphatic: The term “heteroaliphatic” refers to an aliphatic groupwherein one or more units selected from C, CH, CH₂, or CH₃ areindependently replaced by a heteroatom. In some embodiments, aheteroaliphatic group is heteroalkyl. In some embodiments, aheteroaliphatic group is heteroalkenyl.

Heteroaryl: The terms “heteroaryl” and “heteroar,” used alone or as partof a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar,” as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3b]-1,4-oxazin-3(4H)one. Aheteroaryl group may be mono or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

Heteroatom: The term “heteroatom” means one or more of oxygen, sulfur,nitrogen, phosphorus, boron, selenium, or silicon (including, anyoxidized form of nitrogen, boron, selenium, sulfur, phosphorus, orsilicon; the quaternized form of any basic nitrogen or; a substitutablenitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl)).

Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,”“heterocyclic radical,” and “heterocyclic ring” are used interchangeablyand refer to a stable 3- to 7 membered monocyclic or 7-10 memberedbicyclic heterocyclic moiety that is either saturated or partiallyunsaturated, and having, in addition to carbon atoms, one or more,preferably one to four, heteroatoms, as defined above. When used inreference to a ring atom of a heterocycle, the term “nitrogen” includesa substituted nitrogen. As an example, in a saturated or partiallyunsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur ornitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (asin pyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Intraperitoneal: The phrases “intraperitoneal administration” and“administered intraperitonealy” as used herein have their art-understoodmeaning referring to administration of a compound or composition intothe peritoneum of a subject.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within an organism (e.g.,animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, and/or microbe).

Lower alkyl: The term “lower alkyl” refers to a C₁₋₄ straight orbranched alkyl group. Example lower alkyl groups are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, and tert-butyl.

Lower haloalkyl: The term “lower haloalkyl” refers to a C₁₋₄ straight orbranched alkyl group that is substituted with one or more halogen atoms.

Optionally substituted: As described herein, compounds of the disclosuremay contain “optionally substituted” moieties. In general, the term“substituted,” whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis disclosure are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(RO)C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘)2; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(°) 3; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR, —SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘);—(CH₂)₀₋₄C(O)NR^(∘)2; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘),—(CH₂)₀₋₄OC(O)NR^(∘) ₂; —C(O)N(OR)R^(∘); —C(O)C(O)R^(∘);—C(O)CH₂C(O)R^(∘); —C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘);—(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂₀R^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘);—S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄ S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂;—N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘);—P(O)R^(∘) ₂; —OP(O)R^(∘) ₂; —OP(O)(OR^(∘))₂; —SiR^(∘) ₃; —(C₁₋₄straight or branched alkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight orbranched alkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substitutedas defined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a 3-12membered saturated, partially unsaturated, or aryl mono- or bicyclicring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(∘) ₃, —OSiR^(∘)3, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(°) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*2))₂₋₃O—, or—S(C(R*2))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6 memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12 membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6 membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Oral: The phrases “oral administration” and “administered orally” asused herein have their art-understood meaning referring toadministration by mouth of a compound or composition.

Parenteral: The phrases “parenteral administration” and “administeredparenterally” as used herein have their art-understood meaning referringto modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal, and intrasternal injection and infusion.

Partially unsaturated: As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Pharmaceutically acceptable salt: The term “pharmaceutically acceptablesalt”, as used herein, refers to salts of such compounds that areappropriate for use in pharmaceutical contexts, i.e., salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge, et al. describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 66: 1-19 (1977). In some embodiments, pharmaceuticallyacceptable salt include, but are not limited to, nontoxic acid additionsalts, which are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. In someembodiments, pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. In someembodiments, pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,sulfonate and aryl sulfonate.

Prodrug: A general, a “prodrug,” as that term is used herein and as isunderstood in the art, is an entity that, when administered to anorganism, is metabolized in the body to deliver an active (e.g.,therapeutic or diagnostic) agent of interest. Typically, such metabolisminvolves removal of at least one “prodrug moiety” so that the activeagent is formed. Various forms of “prodrugs” are known in the art. Forexamples of such prodrug moieties, see:

-   -   a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985)        and Methods in Enzymology, 42:309-396, edited by K. Widder, et        al. (Academic Press, 1985);    -   b) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley,        2011);    -   c) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley,        2011);    -   d) A Textbook of Drug Design and Development, edited by        Krogsgaard-Larsen;    -   e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”,        by H. Bundgaard, p. 113-191 (1991);    -   f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992);    -   g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285        (1988); and    -   h) Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984).

As with other compounds described herein, prodrugs may be provided inany of a variety of forms, e.g., crystal forms, salt forms etc. In someembodiments, prodrugs are provided as pharmaceutically acceptable saltsthereof.

Protecting group: The term “protecting group,” as used herein, is wellknown in the art and includes those described in detail in ProtectingGroups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd)edition, John Wiley & Sons, 1999, the entirety of which is incorporatedherein by reference. Also included are those protecting groups speciallyadapted for nucleoside and nucleotide chemistry described in CurrentProtocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al.06/2012, the entirety of Chapter 2 is incorporated herein by reference.Suitable amino-protecting groups include methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methyl sulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), 0-trimethyl silylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Suitably protected carboxylic acids further include, but are not limitedto, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylicacids. Examples of suitable silyl groups include trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,triisopropylsilyl, and the like. Examples of suitable alkyl groupsinclude methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl,t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groupsinclude allyl. Examples of suitable aryl groups include optionallysubstituted phenyl, biphenyl, or naphthyl. Examples of suitablearylalkyl groups include optionally substituted benzyl (e.g.,p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, 0-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2-and 4-picolyl.

Suitable hydroxyl protecting groups include methyl, methoxylmethyl(MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethyl silylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4‘’-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl,9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

In some embodiments, a hydroxyl protecting group is acetyl, t-butyl,t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,1-(2-chloroethoxy)ethyl, 2-trimethyl silylethyl, p-chlorophenyl,2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl,diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl),4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl,trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate,tosylate, triflate, trityl, monomethoxytrityl (MMTr),4,4′-dimethoxytrityl, (DMTr) and 4,4′,4″-trimethoxytrityl (TMTr),2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE),2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl(NPE), 2-(4-nitrophenyl sulfonyl)ethyl, 3,5-dichlorophenyl,2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl,2-(2-nitrophenyl)ethyl, butylthiocarbonyl,4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl,2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl(Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl(MOX). In some embodiments, each of the hydroxyl protecting groups is,independently selected from acetyl, benzyl, t-butyldimethylsilyl,t-butyldiphenylsilyl and 4,4′-dimethoxytrityl. In some embodiments, thehydroxyl protecting group is selected from the group consisting oftrityl, monomethoxytrityl and 4,4′-dimethoxytrityl group.

In some embodiments, a phosphorus protecting group is a group attachedto the internucleotide phosphorus linkage throughout oligonucleotidesynthesis. In some embodiments, the phosphorus protecting group isattached to the sulfur atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorus protecting group isattached to the oxygen atom of the internucleotide phosphorothioatelinkage. In some embodiments, the phosphorus protecting group isattached to the oxygen atom of the internucleotide phosphate linkage. Insome embodiments the phosphorus protecting group is 2-cyanoethyl (CE orCne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl,benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe),2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl,4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl,3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl,2-(N-formyl,N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least two amino acids linked to one another bypeptide bonds). In some embodiments, proteins include onlynaturally-occurring amino acids. In some embodiments, proteins includeone or more non-naturally-occurring amino acids (e.g., moieties thatform one or more peptide bonds with adjacent amino acids). In someembodiments, one or more residues in a protein chain contain anon-amino-acid moiety (e.g., a glycan, etc). In some embodiments, aprotein includes more than one polypeptide chain, for example linked byone or more disulfide bonds or associated by other means. In someembodiments, proteins contain L-amino acids, D-amino acids, or both; insome embodiments, proteins contain one or more amino acid modificationsor analogs known in the art. Useful modifications include, e.g.,terminal acetylation, amidation, methylation, etc. The term “peptide” isgenerally used to refer to a polypeptide having a length of less thanabout 100 amino acids, less than about 50 amino acids, less than 20amino acids, or less than 10 amino acids. In some embodiments, proteinsare antibodies, antibody fragments, biologically active portionsthereof, and/or characteristic portions thereof.

Sample: A “sample” as used herein is a specific organism or materialobtained therefrom. In some embodiments, a sample is a biological sampleobtained or derived from a source of interest, as described herein. Insome embodiments, a source of interest comprises an organism, such as ananimal or human. In some embodiments, a biological sample comprisesbiological tissue or fluid. In some embodiments, a biological sample isor comprises bone marrow; blood; blood cells; ascites; tissue or fineneedle biopsy samples; cell-containing body fluids; free floatingnucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritonealfluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs;vaginal swabs; oral swabs; nasal swabs; washings or lavages such as aductal lavages or broncheoalveolar lavages; aspirates; scrapings; bonemarrow specimens; tissue biopsy specimens; surgical specimens; feces,other body fluids, secretions, and/or excretions; and/or cellstherefrom, etc. In some embodiments, a biological sample is or comprisescells obtained from an individual. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by anyappropriate means. For example, in some embodiments, a primarybiological sample is obtained by methods selected from the groupconsisting of biopsy (e.g., fine needle aspiration or tissue biopsy),surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.In some embodiments, as will be clear from context, the term “sample”refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or more agentsto) a primary sample. For example, filtering using a semi-permeablemembrane. Such a “processed sample” may comprise, for example nucleicacids or proteins extracted from a sample or obtained by subjecting aprimary sample to techniques such as amplification or reversetranscription of mRNA, isolation and/or purification of certaincomponents, etc. In some embodiments, a sample is an organism. In someembodiments, a sample is a plant. In some embodiments, a sample is ananimal. In some embodiments, a sample is a human. In some embodiments, asample is an organism other than a human.

Stereochemically isomeric forms: The phrase “stereochemically isomericforms,” as used herein, refers to different compounds made up of thesame atoms bonded by the same sequence of bonds but having differentthree-dimensional structures which are not interchangeable. In someembodiments of the disclosure, provided chemical compositions may be orinclude pure preparations of individual stereochemically isomeric formsof a compound; in some embodiments, provided chemical compositions maybe or include mixtures of two or more stereochemically isomeric forms ofthe compound. In certain embodiments, such mixtures contain equalamounts of different stereochemically isomeric forms; in certainembodiments, such mixtures contain different amounts of at least twodifferent stereochemically isomeric forms. In some embodiments, achemical composition may contain all diastereomers and/or enantiomers ofthe compound. In some embodiments, a chemical composition may containless than all diastereomers and/or enantiomers of a compound. In someembodiments, if a particular enantiomer of a compound of the presentdisclosure is desired, it may be prepared, for example, by asymmetricsynthesis, or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, diastereomeric saltsare formed with an appropriate optically-active acid, and resolved, forexample, by fractional crystallization.

Subject: As used herein, the term “subject” or “test subject” refers toany organism to which a provided compound or composition is administeredin accordance with the present disclosure e.g., for experimental,diagnostic, prophylactic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans; insects; worms; etc.) and plants. In someembodiments, a subject may be suffering from, and/or susceptible to adisease, disorder, and/or condition.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and/or chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with and/or displays oneor more symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition is one who has a higher risk of developingthe disease, disorder, and/or condition than does a member of thegeneral public. In some embodiments, an individual who is susceptible toa disease, disorder and/or condition may not have been diagnosed withthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionmay exhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

Systemic: The phrases “systemic administration,” “administeredsystemically,” “peripheral administration,” and “administeredperipherally” as used herein have their art-understood meaning referringto administration of a compound or composition such that it enters therecipient's system.

Tautomeric forms: The phrase “tautomeric forms,” as used herein, is usedto describe different isomeric forms of organic compounds that arecapable of facile interconversion. Tautomers may be characterized by theformal migration of a hydrogen atom or proton, accompanied by a switchof a single bond and adjacent double bond. In some embodiments,tautomers may result from prototropic tautomerism (i.e., the relocationof a proton). In some embodiments, tautomers may result from valencetautomerism (i.e., the rapid reorganization of bonding electrons). Allsuch tautomeric forms are intended to be included within the scope ofthe present disclosure. In some embodiments, tautomeric forms of acompound exist in mobile equilibrium with each other, so that attemptsto prepare the separate substances results in the formation of amixture. In some embodiments, tautomeric forms of a compound areseparable and isolatable compounds. In some embodiments of thedisclosure, chemical compositions may be provided that are or includepure preparations of a single tautomeric form of a compound. In someembodiments of the disclosure, chemical compositions may be provided asmixtures of two or more tautomeric forms of a compound. In certainembodiments, such mixtures contain equal amounts of different tautomericforms; in certain embodiments, such mixtures contain different amountsof at least two different tautomeric forms of a compound. In someembodiments of the disclosure, chemical compositions may contain alltautomeric forms of a compound. In some embodiments of the disclosure,chemical compositions may contain less than all tautomeric forms of acompound. In some embodiments of the disclosure, chemical compositionsmay contain one or more tautomeric forms of a compound in amounts thatvary over time as a result of interconversion. In some embodiments ofthe disclosure, the tautomerism is keto-enol tautomerism. One of skillin the chemical arts would recognize that a keto-enol tautomer can be“trapped” (i.e., chemically modified such that it remains in the “enol”form) using any suitable reagent known in the chemical arts in toprovide an enol derivative that may subsequently be isolated using oneor more suitable techniques known in the art. Unless otherwiseindicated, the present disclosure encompasses all tautomeric forms ofrelevant compounds, whether in pure form or in admixture with oneanother.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect. In some embodiments, a therapeutic agent is any substance thatcan be used to alleviate, ameliorate, relieve, inhibit, prevent, delayonset of, reduce severity of, and/or reduce incidence of one or moresymptoms or features of a disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a substance (e.g.,a therapeutic agent, composition, and/or formulation) that elicits adesired biological response when administered as part of a therapeuticregimen. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, diagnose, prevent, and/or delay the onset of thedisease, disorder, and/or condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount.

Treat: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof, and/or reduce incidence of one or more symptoms or features of adisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the disease, disorder, and/orcondition, for example for the purpose of decreasing the risk ofdeveloping pathology associated with the disease, disorder, and/orcondition.

Unsaturated: The term “unsaturated,” as used herein, means that a moietyhas one or more units of unsaturation.

Unit dose: The expression “unit dose” as used herein refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

Wild-type: As used herein, the term “wild-type” has its art-understoodmeaning that refers to an entity having a structure and/or activity asfound in nature in a “normal” (as contrasted with mutant, diseased,altered, etc) state or context. Those of ordinary skill in the art willappreciate that wild type genes and polypeptides often exist in multipledifferent forms (e.g., alleles).

Nucleic acid: The term “nucleic acid” includes any nucleotides, analogsthereof, and polymers thereof. The term “polynucleotide” as used hereinrefer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecules and, thus, include double- andsingle-stranded DNA, and double- and single-stranded RNA. These termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated, protected and/or capped nucleotides orpolynucleotides. The terms encompass poly- or oligo-ribonucleotides(RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derivedfrom N-glycosides or C-glycosides of nucleobases and/or modifiednucleobases; nucleic acids derived from sugars and/or modified sugars;and nucleic acids derived from phosphate bridges and/or modifiedphosphorus-atom bridges (also referred to herein as “internucleotidelinkages”). The term encompasses nucleic acids containing anycombinations of nucleobases, modified nucleobases, sugars, modifiedsugars, phosphate bridges or modified phosphorus atom bridges. Examplesinclude, and are not limited to, nucleic acids containing ribosemoieties, the nucleic acids containing deoxy-ribose moieties, nucleicacids containing both ribose and deoxyribose moieties, nucleic acidscontaining ribose and modified ribose moieties. The prefix poly- refersto a nucleic acid containing 2 to about 10,000 nucleotide monomer unitsand wherein the prefix oligo- refers to a nucleic acid containing 2 toabout 200 nucleotide monomer units.

Nucleotide: The term “nucleotide” as used herein refers to a monomericunit of a polynucleotide that consists of a heterocyclic base, a sugar,and one or more phosphate groups or phosphorus-containinginternucleotidic linkages. The naturally occurring bases, (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) arederivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleotides are linked via internucleotidiclinkages to form nucleic acids, or polynucleotides. Manyinternucleotidic linkages are known in the art (such as, though notlimited to, phosphate, phosphorothioates, boranophosphates and thelike). Artificial nucleic acids include PNAs (peptide nucleic acids),phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates,boranophosphates, methylphosphonates, phosphonoacetates,thiophosphonoacetates and other variants of the phosphate backbone ofnative nucleic acids, such as those described herein.

Nucleoside: The term “nucleoside” refers to a moiety wherein anucleobase or a modified nucleobase is covalently bound to a sugar ormodified sugar.

Sugar: The term “sugar” refers to a monosaccharide in closed and/or openform. Sugars include, but are not limited to, ribose, deoxyribose,pentofuranose, pentopyranose, and hexopyranose moieties. As used herein,the term also encompasses structural analogs used in lieu ofconventional sugar molecules, such as glycol, polymer of which forms thebackbone of the nucleic acid analog, glycol nucleic acid (“GNA”).

Modified sugar: The term “modified sugar” refers to a moiety that canreplace a sugar. The modified sugar mimics the spatial arrangement,electronic properties, or some other physicochemical property of asugar.

Nucleobase: The term “nucleobase” refers to the parts of nucleic acidsthat are involved in the hydrogen-bonding that binds one nucleic acidstrand to another complementary strand in a sequence specific manner.The most common naturally-occurring nucleobases are adenine (A), guanine(G), uracil (U), cytosine (C), and thymine (T). In some embodiments, thenaturally-occurring nucleobases are modified adenine, guanine, uracil,cytosine, or thymine. In some embodiments, the naturally-occurringnucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, a nucleobase is a “modified nucleobase,”e.g., a nucleobase other than adenine (A), guanine (G), uracil (U),cytosine (C), and thymine (T). In some embodiments, the modifiednucleobases are methylated adenine, guanine, uracil, cytosine, orthymine. In some embodiments, the modified nucleobase mimics the spatialarrangement, electronic properties, or some other physicochemicalproperty of the nucleobase and retains the property of hydrogen-bondingthat binds one nucleic acid strand to another in a sequence specificmanner. In some embodiments, a modified nucleobase can pair with all ofthe five naturally occurring bases (uracil, thymine, adenine, cytosine,or guanine) without substantially affecting the melting behavior,recognition by intracellular enzymes or activity of the oligonucleotideduplex.

Chiral ligand: The term “chiral ligand” or “chiral auxiliary” refers toa moiety that is chiral and can be incorporated into a reaction so thatthe reaction can be carried out with certain stereoselectivity.

Condensing reagent: In a condensation reaction, the term “condensingreagent” refers to a reagent that activates a less reactive site andrenders it more susceptible to attack by another reagent. In someembodiments, such another reagent is a nucleophile.

Blocking group: The term “blocking group” refers to a group that masksthe reactivity of a functional group. The functional group can besubsequently unmasked by removal of the blocking group. In someembodiments, a blocking group is a protecting group.

Moiety: The term “moiety” refers to a specific segment or functionalgroup of a molecule. Chemical moieties are often recognized chemicalentities embedded in or appended to a molecule.

Solid support: The term “solid support” refers to any support whichenables synthesis of nucleic acids. In some embodiments, the term refersto a glass or a polymer, that is insoluble in the media employed in thereaction steps performed to synthesize nucleic acids, and is derivatizedto comprise reactive groups. In some embodiments, the solid support isHighly Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). Insome embodiments, the solid support is Controlled Pore Glass (CPG). Insome embodiments, the solid support is hybrid support of Controlled PoreGlass (CPG) and Highly Cross-linked Polystyrene (HCP).

Linking moiety: The term “linking moiety” refers to any moietyoptionally positioned between the terminal nucleoside and the solidsupport or between the terminal nucleoside and another nucleoside,nucleotide, or nucleic acid.

DNA molecule: A “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form or a double-stranded helix. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences can be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

Coding sequence: A DNA “coding sequence” or “coding region” is adouble-stranded DNA sequence which is transcribed and translated into apolypeptide in vivo when placed under the control of appropriateexpression control sequences. The boundaries of the coding sequence (the“open reading frame” or “ORF”) are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequencesfrom eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence is,usually, be located 3′ to the coding sequence. The term “non-codingsequence” or “non-coding region” refers to regions of a polynucleotidesequence that are not translated into amino acids (e.g. 5′ and 3′un-translated regions).

Reading frame: The term “reading frame” refers to one of the sixpossible reading frames, three in each direction, of the double strandedDNA molecule. The reading frame that is used determines which codons areused to encode amino acids within the coding sequence of a DNA molecule.

Antisense: As used herein, an “antisense” nucleic acid moleculecomprises a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule, complementary to an mRNAsequence or complementary to the coding strand of a gene. Accordingly,an antisense nucleic acid molecule can associate via hydrogen bonds to asense nucleic acid molecule.

Wobble position: As used herein, a “wobble position” refers to the thirdposition of a codon. Mutations in a DNA molecule within the wobbleposition of a codon, in some embodiments, result in silent orconservative mutations at the amino acid level. For example, there arefour codons that encode Glycine, i.e., GGU, GGC, GGA and GGG, thusmutation of any wobble position nucleotide, to any other nucleotideselected from A, U, C and G, does not result in a change at the aminoacid level of the encoded protein and, therefore, is a silentsubstitution.

Silent substitution: a “silent substitution” or “silent mutation” is onein which a nucleotide within a codon is modified, but does not result ina change in the amino acid residue encoded by the codon. Examplesinclude mutations in the third position of a codon, as well in the firstposition of certain codons such as in the codon “CGG” which, whenmutated to AGG, still encodes Arg.

Gene: The terms “gene,” “recombinant gene” and “gene construct” as usedherein, refer to a DNA molecule, or portion of a DNA molecule, thatencodes a protein or a portion thereof. The DNA molecule can contain anopen reading frame encoding the protein (as exon sequences) and canfurther include intron sequences. The term “intron” as used herein,refers to a DNA sequence present in a given gene which is not translatedinto protein and is found in some, but not all cases, between exons. Itcan be desirable for the gene to be operably linked to, (or it cancomprise), one or more promoters, enhancers, repressors and/or otherregulatory sequences to modulate the activity or expression of the gene,as is well known in the art.

Complementary DNA: As used herein, a “complementary DNA” or “cDNA”includes recombinant polynucleotides synthesized by reversetranscription of mRNA and from which intervening sequences (introns)have been removed.

Homology: “Homology” or “identity” or “similarity” refers to sequencesimilarity between two nucleic acid molecules. Homology and identity caneach be determined by comparing a position in each sequence which can bealigned for purposes of comparison. When an equivalent position in thecompared sequences is occupied by the same base, then the molecules areidentical at that position; when the equivalent site occupied by thesame or a similar nucleic acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage ofhomology/similarity or identity refers to a function of the number ofidentical or similar nucleic acids at positions shared by the comparedsequences. A sequence which is “unrelated” or “non-homologous” sharesless than 40% identity, less than 35% identity, less than 30% identity,or less than 25% identity with a sequence described herein. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

In some embodiments, the term “homology” describes a mathematicallybased comparison of sequence similarities which is used to identifygenes with similar functions or motifs. The nucleic acid sequencesdescribed herein can be used as a “query sequence” to perform a searchagainst public databases, for example, to identify other family members,related sequences or homologs. In some embodiments, such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments,BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleotide sequences homologous tonucleic acid molecules of the disclosure. In some embodiments, to obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used (See www.ncbi.nlm.nih.gov).

Identity: As used herein, “identity” means the percentage of identicalnucleotide residues at corresponding positions in two or more sequenceswhen the sequences are aligned to maximize sequence matching, i.e.,taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well-known Smith Watermanalgorithm can also be used to determine identity.

Heterologous: A “heterologous” region of a DNA sequence is anidentifiable segment of DNA within a larger DNA sequence that is notfound in association with the larger sequence in nature. Thus, when theheterologous region encodes a mammalian gene, the gene can usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous codingsequence is a sequence where the coding sequence itself is not found innature (e.g., a cDNA where the genomic coding sequence contains intronsor synthetic sequences having codons or motifs different than theunmodified gene). Allelic variations or naturally-occurring mutationalevents do not give rise to a heterologous region of DNA as definedherein.

Transition mutation: The term “transition mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by another pyrimidine, or a purine (adenosine(A) or guanosine (G) is replaced by another purine.

Transversion mutation: The term “transversion mutations” refers to basechanges in a DNA sequence in which a pyrimidine (cytidine (C) orthymidine (T) is replaced by a purine (adenosine (A) or guanosine (G),or a purine is replaced by a pyrimidine.

Oligonucleotide: the term “oligonucleotide” refers to a polymer oroligomer of nucleotide monomers, containing any combination ofnucleobases, modified nucleobases, sugars, modified sugars, phosphatebridges, or modified phosphorus atom bridges (also referred to herein as“internucleotidic linkage”, defined further herein).

Oligonucleotides can be single-stranded or double-stranded. As usedherein, the term “oligonucleotide strand” encompasses a single-strandedoligonucleotide. A single-stranded oligonucleotide can havedouble-stranded regions and a double-stranded oligonucleotide can havesingle-stranded regions. Example oligonucleotides include, but are notlimited to structural genes, genes including control and terminationregions, self-replicating systems such as viral or plasmid DNA,single-stranded and double-stranded siRNAs and other RNA interferencereagents (RNAi agents or iRNA agents), shRNA, antisenseoligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, U1 adaptors, triplex-formingoligonucleotides, G-quadruplex oligonucleotides, RNA activators,immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

Double-stranded and single-stranded oligonucleotides that are effectivein inducing RNA interference are also referred to as siRNA, RNAi agent,or iRNA agent, herein. In some embodiments, these RNA interferenceinducing oligonucleotides associate with a cytoplasmic multi-proteincomplex known as RNAi-induced silencing complex (RISC). In manyembodiments, single-stranded and double-stranded RNAi agents aresufficiently long that they can be cleaved by an endogenous molecule,e.g., by Dicer, to produce smaller oligonucleotides that can enter theRISC machinery and participate in RISC mediated cleavage of a targetsequence, e.g. a target mRNA.

Oligonucleotides of the present disclosure can be of various lengths. Inparticular embodiments, oligonucleotides can range from about 2 to about200 nucleotides in length. In various related embodiments,oligonucleotides, single-stranded, double-stranded, and triple-stranded,can range in length from about 4 to about 10 nucleotides, from about 10to about 50 nucleotides, from about 20 to about 50 nucleotides, fromabout 15 to about 30 nucleotides, from about 20 to about 30 nucleotidesin length. In some embodiments, the oligonucleotide is from about 9 toabout 39 nucleotides in length. In some embodiments, the oligonucleotideis at least 4 nucleotides in length. In some embodiments, theoligonucleotide is at least 5 nucleotides in length. In someembodiments, the oligonucleotide is at least 6 nucleotides in length. Insome embodiments, the oligonucleotide is at least 7 nucleotides inlength. In some embodiments, the oligonucleotide is at least 8nucleotides in length. In some embodiments, the oligonucleotide is atleast 9 nucleotides in length. In some embodiments, the oligonucleotideis at least 10 nucleotides in length. In some embodiments, theoligonucleotide is at least 11 nucleotides in length. In someembodiments, the oligonucleotide is at least 12 nucleotides in length.In some embodiments, the oligonucleotide is at least 15 nucleotides inlength. In some embodiments, the oligonucleotide is at least 20nucleotides in length. In some embodiments, the oligonucleotide is atleast 25 nucleotides in length. In some embodiments, the oligonucleotideis at least 30 nucleotides in length. In some embodiments, theoligonucleotide is a duplex of complementary strands of at least 18nucleotides in length. In some embodiments, the oligonucleotide is aduplex of complementary strands of at least 21 nucleotides in length.

Internucleotidic linkage: As used herein, the phrase “internucleotidiclinkage” refers generally to the phosphorus-containing linkage betweennucleotide units of an oligonucleotide, and is interchangeable with“inter-sugar linkage” and “phosphorus atom bridge,” as used above andherein. In some embodiments, an internucleotidic linkage is aphosphodiester linkage, as found in naturally occurring DNA and RNAmolecules. In some embodiments, an internucleotidic linkage is a“modified internucleotidic linkage” wherein each oxygen atom of thephosphodiester linkage is optionally and independently replaced by anorganic or inorganic moiety. In some embodiments, such an organic orinorganic moiety is selected from but not limited to ═S, ═Se, ═NR′,—SR′, —SeR′, —N(R′)₂, B(R′)₃, —S—, —Se—, and —N(R′)—, wherein each R′ isindependently as defined and described below. In some embodiments, aninternucleotidic linkage is a phosphotriester linkage, phosphorothioatediester linkage

or modified phosphorothioate triester linkage. It is understood by aperson of ordinary skill in the art that the internucleotidic linkagemay exist as an anion or cation at a given pH due to the existence ofacid or base moieties in the linkage.

Unless otherwise specified, when used with an oligonucleotide sequence,each of s, s1, s2, s3, s4, s5, s6 and s7 independently represents thefollowing modified internucleotidic linkage as illustrated in Table 1,below.

TABLE 1 Example Modified Internucleotidic Linkage. Symbol ModifiedInternucleotidic Linkage s

s1

s2

s3

s4

s5

s6

s7

s8

s9

s10

s11

s12

s13

s14

s15

s16

s17

s18

For instance, (Rp, Sp)-ATsCs1GA has 1) a phosphorothioateinternucleotidic linkage

between Ta nd C; and 2) a phosphorothioate triester internucleotidiclinkage having the structure of

between C and G. Unless otherwise specified, the Rp/Sp designationspreceding an oligonucleotide sequence describe the configurations ofchiral linkage phosphorus atoms in the internucleotidic linkagessequentially from 5′ to 3′ of the oligonucleotide sequence. Forinstance, in (Rp, Sp)-ATsCs1GA, the phosphorus in the “s” linkagebetween T and C has Rp configuration and the phosphorus in “s1” linkagebetween C and G has Sp configuration. In some embodiments, “All-(Rp)” or“All-(Sp)” is used to indicate that all chiral linkage phosphorus atomsin oligonucleotide have the same Rp or Sp configuration, respectively.For instance, All-(Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ IDNO: 1) indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Rp configuration;All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 2)indicates that all the chiral linkage phosphorus atoms in theoligonucleotide have Sp configuration.

Oligonucleotide type: As used herein, the phrase “oligonucleotide type”is used to define an oligonucleotide that has a particular basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “-XLR¹” groups in formula I).Oligonucleotides of a common designated “type” are structurallyidentical to one another.

One of skill in the art will appreciate that synthetic methods of thepresent disclosure provide for a degree of control during the synthesisof an oligonucleotide strand such that each nucleotide unit of theoligonucleotide strand can be designed and/or selected in advance tohave a particular stereochemistry at the linkage phosphorus and/or aparticular modification at the linkage phosphorus, and/or a particularbase, and/or a particular sugar. In some embodiments, an oligonucleotidestrand is designed and/or selected in advance to have a particularcombination of stereocenters at the linkage phosphorus. In someembodiments, an oligonucleotide strand is designed and/or determined tohave a particular combination of modifications at the linkagephosphorus. In some embodiments, an oligonucleotide strand is designedand/or selected to have a particular combination of bases. In someembodiments, an oligonucleotide strand is designed and/or selected tohave a particular combination of one or more of the above structuralcharacteristics. The present disclosure provides compositions comprisingor consisting of a plurality of oligonucleotide molecules (e.g.,chirally controlled oligonucleotide compositions). In some embodiments,all such molecules are of the same type (i.e., are structurallyidentical to one another). In many embodiments, however, providedcompositions comprise a plurality of oligonucleotides of differenttypes, typically in pre-determined relative amounts.

Chiral control: As used herein, “chiral control” refers to an ability tocontrol the stereochemical designation of every chiral linkagephosphorus within an oligonucleotide strand. The phrase “chirallycontrolled oligonucleotide” refers to an oligonucleotide which exists ina single diastereomeric form with respect to the chiral linkagephosphorus.

Chirally controlled oligonucleotide composition: As used herein, thephrase “chirally controlled oligonucleotide composition” refers to anoligonucleotide composition that contains predetermined levels ofindividual oligonucleotide types. For instance, in some embodiments achirally controlled oligonucleotide composition comprises oneoligonucleotide type. In some embodiments, a chirally controlledoligonucleotide composition comprises more than one oligonucleotidetype. In some embodiments, a chirally controlled oligonucleotidecomposition comprises a mixture of multiple oligonucleotide types.Example chirally controlled oligonucleotide compositions are describedfurther herein.

Chirally pure: as used herein, the phrase “chirally pure” is used todescribe a chirally controlled oligonucleotide composition in which allof the oligonucleotides exist in a single diastereomeric form withrespect to the linkage phosphorus.

Chirally uniform: as used herein, the phrase “chirally uniform” is usedto describe an oligonucleotide molecule or type in which all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, an oligonucleotide whose nucleotide units all have Rpstereochemistry at the linkage phosphorus is chirally uniform. Likewise,an oligonucleotide whose nucleotide units all have Sp stereochemistry atthe linkage phosphorus is chirally uniform.

Predetermined: By predetermined is meant deliberately selected, forexample as opposed to randomly occurring or achieved. Those of ordinaryskill in the art, reading the present specification, will appreciatethat the present disclosure provides new and surprising technologiesthat permit selection of particular oligonucleotide types forpreparation and/or inclusion in provided compositions, and furtherpermits controlled preparation of precisely the selected particulartypes, optionally in selected particular relative amounts, so thatprovided compositions are prepared. Such provided compositions are“predetermined” as described herein. Compositions that may containcertain individual oligonucleotide types because they happen to havebeen generated through a process that cannot be controlled tointentionally generate the particular oligonucleotide types is not a“predetermined” composition. In some embodiments, a predeterminedcomposition is one that can be intentionally reproduced (e.g., throughrepetition of a controlled process).

Linkage phosphorus: as defined herein, the phrase “linkage phosphorus”is used to indicate that the particular phosphorus atom being referredto is the phosphorus atom present in the internucleotidic linkage, whichphosphorus atom corresponds to the phosphorus atom of a phosphodiesterof an internucleotidic linkage as occurs in naturally occurring DNA andRNA. In some embodiments, a linkage phosphorus atom is in a modifiedinternucleotidic linkage, wherein each oxygen atom of a phosphodiesterlinkage is optionally and independently replaced by an organic orinorganic moiety. In some embodiments, a linkage phosphorus atom is P*of formula I. In some embodiments, a linkage phosphorus atom is chiral.In some embodiments, a chiral linkage phosphorus atom is P* of formulaI.

P-modification: as used herein, the term “P-modification” refers to anymodification at the linkage phosphorus other than a stereochemicalmodification. In some embodiments, a P-modification comprises addition,substitution, or removal of a pendant moiety covalently attached to alinkage phosphorus. In some embodiments, the “P-modification” is —X-L-R¹wherein each of X, L and R¹ is independently as defined and describedherein and below.

Blockmer: the term “blockmer,” as used herein, refers to anoligonucleotide strand whose pattern of structural featurescharacterizing each individual nucleotide unit is characterized by thepresence of at least two consecutive nucleotide units sharing a commonstructural feature at the internucleotidic phosphorus linkage. By commonstructural feature is meant common stereochemistry at the linkagephosphorus or a common modification at the linkage phosphorus. In someembodiments, the at least two consecutive nucleotide units sharing acommon structure feature at the internucleotidic phosphours linkage arereferred to as a “block”.

In some embodiments, a blockmer is a “stereoblockmer,” e.g., at leasttwo consecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. Such at lest two consecutive nucleotide units form a“stereoblock.” For instance, (Sp, Sp)-ATsCs1GA is a stereoblockmerbecause at least two consecutive nucleotide units, the Ts and the Cs1,have the same stereochemistry at the linkage phosphorus (both Sp). Inthe same oligonucleotide (Sp, Sp)-ATsCs1GA, TsCs1 forms a block, and itis a stereoblock.

In some embodiments, a blockmer is a “P-modification blockmer,” e.g., atleast two consecutive nucleotide units have the same modification at thelinkage phosphorus. Such at lest two consecutive nucleotide units form a“P-modification block”. For instance, (Rp, Sp)-ATsCsGA is aP-modification blockmer because at least two consecutive nucleotideunits, the Ts and the Cs, have the same P-modification (i.e., both are aphosphorothioate diester). In the same oligonucleotide of (Rp,Sp)-ATsCsGA, TsCs forms a block, and it is a P-modification block.

In some embodiments, a blockmer is a “linkage blockmer,” e.g., at leasttwo consecutive nucleotide units have identical stereochemistry andidentical modifications at the linkage phosphorus. At least twoconsecutive nucleotide units form a “linkage block”. For instance, (Rp,Rp)-ATsCsGA is a linkage blockmer because at least two consecutivenucleotide units, the Ts and the Cs, have the same stereochemistry (bothRp) and P-modification (both phosphorothioate). In the sameoligonucleotide of (Rp, Rp)-ATsCsGA, TsCs forms a block, and it is alinkage block.

In some embodiments, a blockmer comprises one or more blocksindependently selected from a stereoblock, a P-modification block and alinkage block. In some embodiments, a blockmer is a stereoblockmer withrespect to one block, and/or a P-modification blockmer with respect toanother block, and/or a linkage blockmer with respect to yet anotherblock. For instance, (Rp, Rp, Rp, Rp, Rp, Sp, Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG (SEQ ID NO: 3) is a stereoblockmer withrespect to the stereoblock AsTsCsGsAs1 (all Rp at linkage phosphorus) orTs1Cs1Gs1 (all Sp at linkage phosphorus), a P-modification blockmer withrespect to the P-modification block AsTsCsGs (all s linkage) orAs1Ts1Cs1Gs1 (all s1 linkage), or a linkage blockmer with respect to thelinkage block AsTsCsGs (all Rp at linkage phosphorus and all s linkage)or Ts1Cs1Gs1 (all Sp at linkage phosphorus and all s1 linkage).

Altmer: the term “altmer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is characterized in that no two consecutivenucleotide units of the oligonucleotide strand share a particularstructural feature at the internucleotidic phosphorus linkage. In someembodiments, an altmer is designed such that it comprises a repeatingpattern. In some embodiments, an altmer is designed such that it doesnot comprise a repeating pattern.

In some embodiments, an altmer is a “stereoaltmer,” e.g., no twoconsecutive nucleotide units have the same stereochemistry at thelinkage phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 4).

In some embodiments, an altmer is a “P-modification altmer” e.g., no twoconsecutive nucleotide units have the same modification at the linkagephosphorus. For instance, All-(Sp)-CAs1GsT, in which each linkagephosphorus has a different P-modification than the others.

In some embodiments, an altmer is a “linkage altmer,” e.g., no twoconsecutive nucleotide units have identical stereochemistry or identicalmodifications at the linkage phosphorus. For instance, (Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC (SEQ ID NO: 5).

Unimer: the term “unimer,” as used herein, refers to an oligonucleotidestrand whose pattern of structural features characterizing eachindividual nucleotide unit is such that all nucleotide units within thestrand share at least one common structural feature at theinternucleotidic phosphorus linkage. By common structural feature ismeant common stereochemistry at the linkage phosphorus or a commonmodification at the linkage phosphorus.

In some embodiments, a unimer is a “stereounimer,” e.g., all nucleotideunits have the same stereochemistry at the linkage phosphorus. Forinstance, All-(Sp)-CsAs1GsT, in which all the linkages have Spphosphorus.

In some embodiments, a unimer is a “P-modification unimer”, e.g., allnucleotide units have the same modification at the linkage phosphorus.For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO:6), in which all the internucleotidic linkages are phosphorothioatediester.

In some embodiments, a unimer is a “linkage unimer,” e.g., allnucleotide units have the same stereochemistry and the samemodifications at the linkage phosphorus. For instance,All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC (SEQ ID NO: 7), inwhich all the internucleotidic linkages are phosphorothioate diesterhaving Sp linkage phosphorus.

Gapmer: as used herein, the term “gapmer” refers to an oligonucleotidestrand characterized in that at least one internucleotidic phosphoruslinkage of the oligonucleotide strand is a phosphate diester linkage,for example such as those found in naturally occurring DNA or RNA. Insome embodiments, more than one internucleotidic phosphorus linkage ofthe oligonucleotide strand is a phosphate diester linkage such as thosefound in naturally occurring DNA or RNA. For instance, All-(Sp)-CAs1GsT,in which the internucleotidic linkage between C and A is a phosphatediester linkage.

Skipmer: as used herein, the term “skipmer” refers to a type of gapmerin which every other internucleotidic phosphorus linkage of theoligonucleotide strand is a phosphate diester linkage, for example suchas those found in naturally occurring DNA or RNA, and every otherinternucleotidic phosphorus linkage of the oligonucleotide strand is amodified internucleotidic linkage. For instance,All-(Sp)-AsTCs1GAs2TCs3G.

For purposes of this disclosure, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The methods and structures described herein relating to compounds andcompositions of the disclosure also apply to the pharmaceuticallyacceptable acid or base addition salts and all stereoisomeric forms ofthese compounds and compositions.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Example dose response of C3a complement activation (measured viaC3a) by oligonucleotides targeting human SOD1 in pooled serum (threeindividual cynomolgus monkeys). 40 min incubation; 37° C.

FIG. 2. Example time course of 3Ca complement activation (measured viaC3a) by SOD1 oligonucleotides in pooled serum (three individualcynomolgus monkeys). Oligonucleotide concentration: 330 μg/mL; 37° C.

FIG. 3. Example time course of 3Ca complement activation (measured viaC3a) by oligonucleotides targeting mouse ApoB in pooled serum (threeindividual cynomolgus monkeys). Oligonucleotide concentration: 330μg/mL; 37° C.

FIG. 4. Example time course of 3Ca complement activation (measured viaC3a) by oligonucleotides targeting human HTT in pooled serum (threeindividual cynomolgus monkeys). Oligonucleotide concentration: 330μg/mL; 37° C.

FIG. 5. Example time course of Bb complement activation (measured viaBb) by oligonucleotides targeting human HTT in pooled serum (threeindividual cynomolgus monkeys). Oligonucleotide concentration: 330μg/mL; 37° C.

FIG. 6. Example albumin binding by oligonucleotides targeting human HTT.

FIG. 7. Example albumin binding by oligonucleotides targeting mouse ApoB

FIG. 8. Example albumin binding by oligonucleotides targeting human SOD1

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Synthetic oligonucleotides provide useful molecular tools in a widevariety of applications. For example, oligonucleotides are useful intherapeutic, diagnostic, research, and new nanomaterials applications.The use of naturally occurring nucleic acids (e.g., unmodified DNA orRNA) is limited, for example, by their susceptibility to endo- andexo-nucleases. As such, various synthetic counterparts have beendeveloped to circumvent these shortcomings. These include syntheticoligonucleotides that contain chemical modification, e.g., basemodifications, sugar modifications, backbone modifications, etc., which,among other things, render these molecules less susceptible todegradation and improve other properties of oligonucleotides. Chemicalmodifications may also lead to certain undesired effects, such asincreased toxicities, etc. From a structural point of view,modifications to internucleotide phosphate linkages introduce chirality,and certain properties of oligonucleotides may be affected by theconfigurations of the phosphorus atoms that form the backbone of theoligonucleotides. For example, in vitro studies have shown that theproperties of antisense nucleotides such as binding affinity, sequencespecific binding to the complementary RNA, stability to nucleases areaffected by, inter alia, chirality of the backbone (e.g., theconfigurations of the phosphorus atoms).

Among other things, the present disclosure encompasses the recognitionthat structural elements of oligonucleotides, such as base sequence,chemical modifications (e.g., modifications of sugar, base, and/orinternucleotidic linkages, and patterns thereof), and/or stereochemistry(e.g., stereochemistry of backbone chiral centers (chiralinternucleotidic linkages), and/or patterns thereof), can havesignificant impact on properties, e.g., activities, toxicities, etc., ofoligonucleotides and can be adjusted to modulate oligonucleotideproperties. In some embodiments, oligonucleotide properties can beadjusted by optimizing chemical modifications (modifications of base,sugar, and/or internucleotidic linkage) and/or stereochemistry (patternof backbone chiral centers).

In some embodiments, the present disclosure demonstrates thatoligonucleotide compositions comprising oligonucleotides with controlledstructural elements, e.g., controlled chemical modification and/orcontrolled backbone stereochemistry patterns, provide unexpectedproperties, including but not limited to those described herein. In someembodiments, provided compositions comprising oligonucleotides havingchemical modifications (e.g., base modifications, sugar modification,internucleotidic linkage modifications, etc.) have improved properties,such as lower toxicity, or improved protein binding profile, or improveddelivery, etc. In some embodiments, provided oligonucleotides inprovided compositions, e.g., oligonucleotides of a first plurality,comprise base modifications, sugar modifications, and/orinternucleotidic linkage modifications. In some embodiments, providedoligonucleotides comprise base modifications and sugar modifications. Insome embodiments, provided oligonucleotides comprise base modificationsand internucleotidic linkage modifications. In some embodiments,provided oligonucleotides comprise sugar modifications andinternucleotidic modifications. In some embodiments, providedcompositions comprise base modifications, sugar modifications, andinternucleotidic linkage modifications. Example chemical modifications,such as base modifications, sugar modifications, internucleotidiclinkage modifications, etc. are widely known in the art including butnot limited to those described in this disclosure. In some embodiments,a modified base is substituted A, T, C, G or U. In some embodiments, asugar modification is 2′-modification. In some embodiments, a2′-modification is 2-F modification. In some embodiments, a2′-modification is 2′-OR¹. In some embodiments, a 2′-modification is2′-OR¹, wherein R¹ is optionally substituted alkyl. In some embodiments,a 2′-modification is 2′-OMe. In some embodiments, a 2′-modification is2′-MOE. In some embodiments, a modified sugar moiety is a bridgedbicyclic or polycyclic ring. In some embodiments, a modified sugarmoiety is a bridged bicyclic or polycyclic ring having 5-20 ring atomswherein one or more ring atoms are optionally and independentlyheteroatoms. Example ring structures are widely known in the art, suchas those found in BNA, LNA, etc. In some embodiments, providedoligonucleotides comprise both one or more modified internucleotidiclinkages and one or more natural phosphate linkages. In someembodiments, oligonucleotides comprising both modified internucleotidiclinkage and natural phosphate linkage and compositions thereof provideimproved properties, e.g., activities and toxicities, etc. In someembodiments, a modified internucleotidic linkage is a chiralinternucleotidic linkage. In some embodiments, a modifiedinternucleotidic linkage is a phosphorothioate linkage. In someembodiments, a modified internucleotidic linkage is a substitutedphosphorothioate linkage. Among other things, the present disclosureencompasses the recognition that stereorandom oligonucleotidepreparations contain a plurality of distinct chemical entities thatdiffer from one another, e.g., in the stereochemical structure ofindividual backbone chiral centers within the oligonucleotide chain.Without control of stereochemistry of backbone chiral centers,stereorandom oligonucleotide preparations provide uncontrolledcompositions comprising undetermined levels of oligonucleotidestereoisomers. Even though these stereoisomers may have the same basesequence, they are different chemical entities at least due to theirdifferent backbone stereochemistry, and they can have, as demonstratedherein, different properties, e.g., activities, toxicities, etc. Amongother things, the present disclosure provides new compositions that areor contain particular stereoisomers of oligonucleotides of interest. Insome embodiments, a particular stereoisomer may be defined, for example,by its base sequence, its length, its pattern of backbone linkages, andits pattern of backbone chiral centers. As is understood in the art, insome embodiments, base sequence may refer to the identity and/ormodification status of nucleoside residues (e.g., of sugar and/or basecomponents, relative to standard naturally occurring nucleotides such asadenine, cytosine, guanosine, thymine, and uracil) in an oligonucleotideand/or to the hybridization character (i.e., the ability to hybridizewith particular complementary residues) of such residues. In someembodiments, oligonucleotides in provided compositions comprise sugarmodifications, e.g., 2′-modifications, at e.g., a wing region. In someembodiments, oligonucleotides in provided compositions comprise a regionin the middle, e.g., a core region, that has no sugar modifications. Insome embodiments, the present disclosure provide an oligonucleotidecomposition comprising a predetermined level of oligonucleotides of anindividual oligonucleotide type which are chemically identical, e.g.,they have the same base sequence, the same pattern of nucleosidemodifications (modifications to sugar and base moieties, if any), thesame pattern of backbone chiral centers, and the same pattern ofbackbone phosphorus modifications. The present disclosure demonstrates,among other things, that individual stereoisomers of a particularoligonucleotide can show different stability and/or activity (e.g.,functional and/or toxicity properties) from each other. In someembodiments, property improvements achieved through inclusion and/orlocation of particular chiral structures within an oligonucleotide canbe comparable to, or even better than those achieved through use ofparticular backbone linkages, residue modifications, etc. (e.g., throughuse of certain types of modified phosphates [e.g., phosphorothioate,substituted phosphorothioate, etc.], sugar modifications [e.g.,2′-modifications, etc.], and/or base modifications [e.g., methylation,etc.]). Among other things, the present disclosure recognizes that, insome embodiments, properties (e.g., activities, toxicities, etc.) of anoligonucleotide can be adjusted by optimizing its pattern of backbonechiral centers, optionally in combination with adjustment/optimizationof one or more other features (e.g., linkage pattern, nucleosidemodification pattern, etc.) of the oligonucleotide. As exemplified byvarious examples in the present disclosure, provided chirally controlledoligonucleotide compositions can demonstrate improved properties, suchas lower toxcicity, improved protein binding profile, improved delivery,etc.

In some embodiments, oligonucleotide properties can be adjusted byoptimizing stereochemistry (pattern of backbone chiral centers) andchemical modifications (modifications of base, sugar, and/orinternucleotidic linkage). Among other things, the present disclosuredemonstrates that stereochemistry can further improve properties ofoligonucleotides comprising chemical modifications. In some embodiments,the present disclosure provides oligonucleotide compositions wherein theoligonucleotides comprise nucleoside modifications, chiralinternucleotidic linkages and natural phosphate linkages. For example,WV-1092 comprises 2′-OMe modifications, phosphate and phosphorothioatelinkages in its 5′- and 3′-wing regions, and phosphorothioate linkagesin its core regions.

In some embodiments, the present disclosure provides oligonucleotidecompositions which, unexpectedly, greatly improve properties ofoligonucleotides. In some embodiments, provided oligonucleotidecompositions provides surprisingly low toxicity. In some embodiments,provided oligonucleotide compositions provides surprisingly improvedprotein binding profile. In some embodiments, provided oligonucleotidecompositions provides surprisingly enhanced delivery. In someembodiments, certain property improvement, such as lower toxicity,improved protein binding profile, and/or enhanced delivery, etc., areachieved without sacrificing other properties, e.g., activities,specificity, etc. In some embodiments, provided compositions provideslower toxicity, improved protein binding profile, and/or enhanceddelivery, and improved activity, stability, and/or specificity (e.g.,target-specificity, cleavage site specificity, etc.). Example improvedactivities (e.g., enhanced cleavage rates, increased target-specificity,cleavage site specificity, etc.) include but are not limited to thosedescribed in WO/2014/012081 and WO/2015/107425.

In some embodiments, a pattern of backbone chiral centers providesincreased stability. In some embodiments, a pattern of backbone chiralcenters provides surprisingly increased activity. In some embodiments, apattern of backbone chiral centers provides increased stability andactivity. In some embodiments, a pattern of backbone chiral centersprovides surprisingly low toxicity. In some embodiments, a pattern ofbackbone chiral centers provides surprisingly low immune response. Insome embodiments, a pattern of backbone chiral centers providessurprisingly low complement activation. In some embodiments, a patternof backbone chiral centers provides surprisingly low complementactivation via the alternative pathway. In some embodiments, a patternof backbone chiral centers provides surprisingly improved proteinbinding profile. In some embodiments, a pattern of backbone chiralcenters provides surprisingly increased binding to certain proteins. Insome embodiments, a pattern of backbone chiral centers providessurprisingly enhanced delivery. In some embodiments, a pattern ofbackbone chiral centers comprises or is (Sp)m(Rp)n, (Rp)n(Sp)m,(Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, a pattern ofbackbone chiral centers comprises or is (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or(Sp)t(Rp)n(Sp)m, wherein m>2. In some embodiments, a pattern of backbonechiral centers comprises or is (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or(Sp)t(Rp)n(Sp)m, wherein n is 1, t>1, and m>2. In some embodiments, m>3.In some embodiments, m>4. In some embodiments, a pattern of backbonechiral centers comprises one or more achiral natural phosphate linkages.In some embodiments, a pattern of backbone chiral centers comprises,comprises one or more repeats of, or is (Sp)m(Rp)n, (Rp)n(Sp)m,(Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments describedherein, m is 1-50; and n is 1-10; and t is 1-50. In some embodiments, apattern of backbone chiral centers comprises or is (Sp)m(Rp)n,(Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, apattern of backbone chiral centers comprises or is (Rp)n(Sp)m,(Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, wherein m>2. In some embodiments, apattern of backbone chiral centers is a sequence comprising at least 5,6, 7, 8, 9, or 10 or more consecutive (Sp) positions. In someembodiments, a pattern of backbone chiral centers is a sequencecomprising at least 5 consecutive (Sp) positions. In some embodiments, apattern of backbone chiral centers is a sequence comprising at least 8consecutive (Sp) positions. In some embodiments, a pattern of backbonechiral centers is a sequence comprising at least 10 consecutive (Sp)positions. In some embodiments, a pattern of backbone chiral centers isa sequence consisting of all (Sp) with a single (Rp). In someembodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp) at or adjacent to the positionof a SNP. In some embodiments, a pattern of backbone chiral centers is asequence consisting of all (Sp) with a single (Rp), wherein the moleculehas a wing-core-wing format. In some embodiments, a pattern of backbonechiral centers is a sequence consisting of all (Sp) with a single (Rp),wherein the molecule has a wing-core-wing format, wherein the wing onthe 5′ end is 1-9 nt long, the core is 1-15 nt long, and the wing on the3′ end is 1-9 nt long. In some embodiments, a pattern of backbone chiralcenters is a sequence consisting of all (Sp) with a single (Rp), whereinthe molecule has a wing-core-wing format, wherein the wing on the 5′ endis 5 nt long, the core is 1-15 nt long, and the wing on the 3′ end is 5nt long. In some embodiments, a pattern of backbone chiral centers is asequence consisting of all (Sp) with a single (Rp), wherein the moleculehas a wing-core-wing format, wherein the wing on the 5′ end is 1-9 ntlong, the core is 10 nt long, and the wing on the 3′ end is 1-9 nt long.In some embodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein the wing on the 5′ end is 5 nt long, thecore is 10 nt long, and the wing on the 3′ end is 5 nt long. In someembodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein the wing on the 5′ end is 5 nt long, thecore is 10 nt long, and the wing on the 3′ end is 5 nt long, and atleast one wing comprises a nucleotide with a 2′-OMe modification. Insome embodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein each wing comprises at least onenucleotide with a 2′-OMe modification. In some embodiments, a pattern ofbackbone chiral centers is a sequence consisting of all (Sp) with asingle (Rp), wherein the molecule has a wing-core-wing format, whereineach nucleotide in both wings has a 2′-OMe modification. In someembodiments, a pattern of backbone chiral centers is a sequenceconsisting of all (Sp) with a single (Rp), wherein the molecule has awing-core-wing format, wherein the wing on the 5′ end is 5 nt long, thecore is 10 nt long, and the wing on the 3′ end is 5 nt long, and eachnucleotide in each wing has a 2′-OMe modification. In some embodiments,the oligonucleotide is single-stranded and has a wing-core-wing format,wherein the wing on the 5′ end of the molecule comprises 4 to 8 nt, eachof which has a 2′-OMe modification and wherein the nt at the 5′ end ofthe molecule has a phosphorothioate in the Sp conformation; the corecomprises 8 to 12 nt, each of which is DNA (2′-H), wherein each has aphosphorothioate in the Sp position except one nt which has thephosphorothioate in the Rp position; and wherein the wing on the 3′ endof the molecule comprises 4 to 8 nt, each of which has a 2′-OMemodification, and wherein the nt at the 3′ end of the molecule comprisesa phosphorothioate in the Sp conformation. In some embodiments, theoligonucleotide is single-stranded and has a wing-core-wing format,wherein the wing on the 5′ end of the molecule comprises 6 nt, each ofwhich has a 2′-OMe modification and wherein the nt at the 5′ end of themolecule has a phosphorothioate in the Sp conformation; the corecomprises 10 nt, each of which is DNA (2′-H), wherein each has aphosphorothioate in the Sp position except one nt which has thephosphorothioate in the Rp position; and wherein the wing on the 3′ endof the molecule comprises 6 nt, each of which has a 2′-OMe modification,and wherein the nt at the 3′ end of the molecule comprises aphosphorothioate in the Sp conformation.

In some embodiments, the present disclosure recognizes that chemicalmodifications, such as modifications of nucleosides and internucleotidiclinkages, can provide enhanced properties. In some embodiments, thepresent disclosure demonstrates that combinations of chemicalmodifications and stereochemistry can provide unexpected, greatlyimproved properties (e.g., bioactivity, selectivity, etc.). In someembodiments, chemical combinations, such as modifications of sugars,bases, and/or internucleotidic linkages, are combined withstereochemistry patterns, e.g., (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or(Sp)t(Rp)n(Sp)m, to provide oligonucleotides and compositions thereofwith surprisingly enhanced properties. In some embodiments, a providedoligonucleotide composition is chirally controlled, and comprises acombination of 2′-modification of one or more sugar moieties, one ormore natural phosphate linkages, one or more phosphorothioate linkages,and a stereochemistry pattern of (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or(Sp)t(Rp)n(Sp)m, wherein m>2. In some embodiments, n is 1, t>1, and m>2.In some embodiments, m>3. In some embodiments, m>4.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides, wherein:

oligonucleotides of the first plurality have the same base sequence; and

oligonucleotides of the first plurality comprise one or more modifiedsugar moieties, or comprise one or more natural phosphate linkages andone or more modified internucleotidic linkages.

In some embodiments, oligonucleotides of the first plurality compriseone or more modified sugar moieties. In some embodiments, providedoligonucleotides comprise one or more modified sugar moieties. In someembodiments, provided oligonucleotides comprise 2 or more modified sugarmoieties. In some embodiments, provided oligonucleotides comprise 3 ormore modified sugar moieties. In some embodiments, providedoligonucleotides comprise 4 or more modified sugar moieties. In someembodiments, provided oligonucleotides comprise 5 or more modified sugarmoieties. In some embodiments, provided oligonucleotides comprise 6 ormore modified sugar moieties. In some embodiments, providedoligonucleotides comprise 7 or more modified sugar moieties. In someembodiments, provided oligonucleotides comprise 8 or more modified sugarmoieties. In some embodiments, provided oligonucleotides comprise 9 ormore modified sugar moieties. In some embodiments, providedoligonucleotides comprise 10 or more modified sugar moieties. In someembodiments, provided oligonucleotides comprise 15 or more modifiedsugar moieties. In some embodiments, provided oligonucleotides comprise20 or more modified sugar moieties. In some embodiments, providedoligonucleotides comprise 25 or more modified sugar moieties. In someembodiments, provided oligonucleotides comprise 30 or more modifiedsugar moieties.

In some embodiments, 5% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 10% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 15% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 20% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 25% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 30% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 35% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 40% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 45% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 50% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 55% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 60% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 65% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 70% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 75% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 80% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 85% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, 90% or more of thesugar moieties of provided oligonucleotides are modified. In someembodiments, 95% or more of the sugar moieties of providedoligonucleotides are modified. In some embodiments, each sugar moiety ofprovided oligonucleotides is modified.

In some embodiments, oligonucleotides of the first plurality compriseone or more natural phosphate linkages and one or more modifiedinternucleotidic linkages.

Provided oligonucleotides can comprise various number of naturalphosphate linkages. In some embodiments, provided oligonucleotidescomprise no natural phosphate linkages. In some embodiments, providedoligonucleotides comprise one natural phosphate linkage. In someembodiments, provided oligonucleotides comprise 2 or more naturalphosphate linkages. In some embodiments, provided oligonucleotidescomprise 3 or more natural phosphate linkages. In some embodiments,provided oligonucleotides comprise 4 or more natural phosphate linkages.In some embodiments, provided oligonucleotides comprise 5 or morenatural phosphate linkages. In some embodiments, providedoligonucleotides comprise 6 or more natural phosphate linkages. In someembodiments, provided oligonucleotides comprise 7 or more naturalphosphate linkages. In some embodiments, provided oligonucleotidescomprise 8 or more natural phosphate linkages. In some embodiments,provided oligonucleotides comprise 9 or more natural phosphate linkages.In some embodiments, provided oligonucleotides comprise 10 or morenatural phosphate linkages. In some embodiments, providedoligonucleotides comprise 15 or more natural phosphate linkages. In someembodiments, provided oligonucleotides comprise 20 or more naturalphosphate linkages. In some embodiments, provided oligonucleotidescomprise 25 or more natural phosphate linkages. In some embodiments,provided oligonucleotides comprise 30 or more natural phosphatelinkages.

In some embodiments, 5% or more of the internucleotidic linkages ofprovided oligonucleotides are natural phosphate linkages. In someembodiments, 10% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,15% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,20% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,25% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,30% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,35% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,40% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,45% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,50% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,55% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,60% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,65% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,70% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,75% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,80% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,85% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,90% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,95% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages.

Provided oligonucleotides can comprise various number of modifiedinternucleotidic linkages. In some embodiments, providedoligonucleotides comprise one modified internucleotidic linkage. In someembodiments, provided oligonucleotides comprise 2 or more modifiedinternucleotidic linkages. In some embodiments, providedoligonucleotides comprise 3 or more modified internucleotidic linkages.In some embodiments, provided oligonucleotides comprise 4 or moremodified internucleotidic linkages. In some embodiments, providedoligonucleotides comprise 5 or more modified internucleotidic linkages.In some embodiments, provided oligonucleotides comprise 6 or moremodified internucleotidic linkages. In some embodiments, providedoligonucleotides comprise 7 or more modified internucleotidic linkages.In some embodiments, provided oligonucleotides comprise 8 or moremodified internucleotidic linkages. In some embodiments, providedoligonucleotides comprise 9 or more modified internucleotidic linkages.In some embodiments, provided oligonucleotides comprise 10 or moremodified internucleotidic linkages. In some embodiments, providedoligonucleotides comprise 15 or more modified internucleotidic linkages.In some embodiments, provided oligonucleotides comprise 20 or moremodified internucleotidic linkages. In some embodiments, providedoligonucleotides comprise 25 or more modified internucleotidic linkages.In some embodiments, provided oligonucleotides comprise 30 or moremodified internucleotidic linkages.

In some embodiments, 5% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 10% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 15% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 20% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 25% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 30% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 35% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 40% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 45% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 50% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 55% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 60% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 65% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 70% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 75% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 80% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 85% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 90% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 95% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, each internucleotidic linkage of providedoligonucleotides is a modified internucleotidic linkage.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and optionally one or more natural phosphatelinkages, and the core region independently comprises one or moremodified internucleotidic linkages; or

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and optionally one or more natural phosphatelinkages, and the core region independently comprises one or moremodified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and optionally one or more natural phosphatelinkages, and the core region independently comprises one or moremodified internucleotidic linkages; and

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and one or more natural phosphate linkages,and the core region independently comprises one or more modifiedinternucleotidic linkages; and

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand optionally one or more natural phosphate linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingone or more wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingtwo wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingtwo wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages;

the wing region to the 5′-end of the core region comprises at least onemodified internucleotidic linkage followed by a natural phosphatelinkage in the wing; and

the wing region to the 3′-end of the core region comprises at least onemodified internucleotidic linkage preceded by a natural phosphatelinkage in the wing;

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisinga wing region and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

the wing region has a length of two or more bases, and comprises one ormore modified internucleotidic linkages and one or more naturalphosphate linkages;

the wing region is to the 5′-end of the core region and comprises amodified internucleotidic linkage between the two nucleosides at its3′-end, or the wing region to the 3′-end of the core region andcomprises a modified internucleotidic linkage between the twonucleosides at its 5′-end; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a first plurality of oligonucleotides comprisingtwo wing regions and a core region, wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing region independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages;

the wing region to the 5′-end of the core region comprises a modifiedinternucleotidic linkage between the two nucleosides at its 3′-end;

the wing region to the 3′-end of a core region comprises a modifiedinternucleotidic linkage between the two nucleosides at its 5′-end; andthe core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

An example composition is WV-1497, wherein the core region is*A*A*G*G*G*C*A*C*A*G* (SEQ ID NO: 8), the wing region to the 5′-end ofthe core region is mG*mGmCmAmC, and the wing region to the 3′-end of thecore region is mAmCmUmU*mC. In some embodiments, a wing region comprisesa modified internucleotidic linkage between the two nucleosides at its3′-end. In some embodiments, a wing region to the 5′-end of a coreregion comprises a modified internucleotidic linkage between the twonucleosides at its 3′-end. For example, in WV-1497, mG*mGmCmAmC is awing to the 5′-end of the core region (*A*A*G*G*G*C*A*C*A*G* (SEQ ID NO:8)), and it comprise a modified internucleotidic linkage between the twonucleosides at its 3′-end (mG*mGmCmAmC). In some embodiments, a wingregion comprises a modified internucleotidic linkage between the twonucleosides at its 5′-end. In some embodiments, a wing region to the3′-end of a core region comprises a modified internucleotidic linkagebetween the two nucleosides at its 5′-end. For example, in WV-1497,mAmCmUmU*mC is a wing to the 3′-end of the core region(*A*A*G*G*G*C*A*C*A*G* (SEQ ID NO: 8)), and it comprise a modifiedinternucleotidic linkage between the two nucleosides at its 5′-end(mAmCmUmU*mC).

In some embodiments, oligonucleotides of the first plurality have twowing and one core regions. In some embodiments, the two wing regions areidentical. In some embodiments, the two wing regions are different.

In some embodiments, a wing region comprises 2 or more modifiedinternucleotidic linkages. In some embodiments, a wing region comprises3 or more modified internucleotidic linkages. In some embodiments, awing region comprises 4 or more modified internucleotidic linkages. Insome embodiments, a wing region comprises 5 or more modifiedinternucleotidic linkages. In some embodiments, a wing region comprises6 or more modified internucleotidic linkages. In some embodiments, awing region comprises 7 or more modified internucleotidic linkages. Insome embodiments, a wing region comprises 8 or more modifiedinternucleotidic linkages. In some embodiments, a wing region comprises9 or more modified internucleotidic linkages. In some embodiments, awing region comprises 10 or more modified internucleotidic linkages. Insome embodiments, a wing region comprises 11 or more modifiedinternucleotidic linkages. In some embodiments, a wing region comprises12 or more modified internucleotidic linkages. In some embodiments, awing region comprises 13 or more modified internucleotidic linkages. Insome embodiments, a wing region comprises 14 or more modifiedinternucleotidic linkages. In some embodiments, a wing region comprises15 or more modified internucleotidic linkages. In some embodiments, awing region comprises 2 or consecutive modified internucleotidiclinkages. In some embodiments, a wing region comprises 3 or consecutivemodified internucleotidic linkages. In some embodiments, a wing regioncomprises 4 or consecutive modified internucleotidic linkages. In someembodiments, a wing region comprises 5 or consecutive modifiedinternucleotidic linkages. In some embodiments, a wing region comprises6 or consecutive modified internucleotidic linkages. In someembodiments, a wing region comprises 7 or consecutive modifiedinternucleotidic linkages. In some embodiments, a wing region comprises8 or consecutive modified internucleotidic linkages. In someembodiments, a wing region comprises 9 or consecutive modifiedinternucleotidic linkages. In some embodiments, a wing region comprises10 or consecutive modified internucleotidic linkages. In someembodiments, a wing region comprises 11 or consecutive modifiedinternucleotidic linkages. In some embodiments, a wing region comprises12 or consecutive modified internucleotidic linkages. In someembodiments, a wing region comprises 13 or consecutive modifiedinternucleotidic linkages. In some embodiments, a wing region comprises14 or consecutive modified internucleotidic linkages. In someembodiments, a wing region comprises 15 or consecutive modifiedinternucleotidic linkages. In some embodiments, each internucleotidiclinkage in a wing region is independently a modified internucleotidiclinkage.

In some embodiments, 5% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 10% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 15% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 20% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 25% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 30% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 35% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 40% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 45% or more of the internucleotidic linkages of a wingregion are modified internucleotidic linkages. In some embodiments, 50%or more of the internucleotidic linkages of a wing region are modifiedinternucleotidic linkages. In some embodiments, 55% or more of theinternucleotidic linkages of a wing region are modified internucleotidiclinkages. In some embodiments, 60% or more of the internucleotidiclinkages of a wing region are modified internucleotidic linkages. Insome embodiments, 65% or more of the internucleotidic linkages of a wingregion are modified internucleotidic linkages. In some embodiments, 70%or more of the internucleotidic linkages of a wing region are modifiedinternucleotidic linkages. In some embodiments, 75% or more of theinternucleotidic linkages of a wing region are modified internucleotidiclinkages. In some embodiments, 80% or more of the internucleotidiclinkages of a wing region are modified internucleotidic linkages. Insome embodiments, 85% or more of the internucleotidic linkages of a wingregion are modified internucleotidic linkages. In some embodiments, 90%or more of the internucleotidic linkages of a wing region are modifiedinternucleotidic linkages. In some embodiments, 95% or more of theinternucleotidic linkages of a wing region are modified internucleotidiclinkages. In some embodiments, each internucleotidic linkage of a wingregion is a modified internucleotidic linkage.

In some embodiments, a wing region comprises 2 or more natural phosphatelinkages. In some embodiments, a wing region comprises 3 or more naturalphosphate linkages. In some embodiments, a wing region comprises 4 ormore natural phosphate linkages. In some embodiments, a wing regioncomprises 5 or more natural phosphate linkages. In some embodiments, awing region comprises 6 or more natural phosphate linkages. In someembodiments, a wing region comprises 7 or more natural phosphatelinkages. In some embodiments, a wing region comprises 8 or more naturalphosphate linkages. In some embodiments, a wing region comprises 9 ormore natural phosphate linkages. In some embodiments, a wing regioncomprises 10 or more natural phosphate linkages. In some embodiments, awing region comprises 11 or more natural phosphate linkages. In someembodiments, a wing region comprises 12 or more natural phosphatelinkages. In some embodiments, a wing region comprises 13 or morenatural phosphate linkages. In some embodiments, a wing region comprises14 or more natural phosphate linkages. In some embodiments, a wingregion comprises 15 or more natural phosphate linkages. In someembodiments, a wing region comprises 2 or consecutive natural phosphatelinkages. In some embodiments, a wing region comprises 3 or consecutivenatural phosphate linkages. In some embodiments, a wing region comprises4 or consecutive natural phosphate linkages. In some embodiments, a wingregion comprises 5 or consecutive natural phosphate linkages. In someembodiments, a wing region comprises 6 or consecutive natural phosphatelinkages. In some embodiments, a wing region comprises 7 or consecutivenatural phosphate linkages. In some embodiments, a wing region comprises8 or consecutive natural phosphate linkages. In some embodiments, a wingregion comprises 9 or consecutive natural phosphate linkages. In someembodiments, a wing region comprises 10 or consecutive natural phosphatelinkages. In some embodiments, a wing region comprises 11 or consecutivenatural phosphate linkages. In some embodiments, a wing region comprises12 or consecutive natural phosphate linkages. In some embodiments, awing region comprises 13 or consecutive natural phosphate linkages. Insome embodiments, a wing region comprises 14 or consecutive naturalphosphate linkages. In some embodiments, a wing region comprises 15 orconsecutive natural phosphate linkages. In some embodiments, eachinternucleotidic linkage in a wing region is independently a naturalphosphate linkage.

In some embodiments, 5% or more of the internucleotidic linkages ofprovided oligonucleotides are natural phosphate linkages. In someembodiments, 10% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,15% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,20% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,25% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,30% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,35% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,40% or more of the internucleotidic linkages of providedoligonucleotides are natural phosphate linkages. In some embodiments,45% or more of the internucleotidic linkages of a wing region arenatural phosphate linkages. In some embodiments, 50% or more of theinternucleotidic linkages of a wing region are natural phosphatelinkages. In some embodiments, 55% or more of the internucleotidiclinkages of a wing region are natural phosphate linkages. In someembodiments, 60% or more of the internucleotidic linkages of a wingregion are natural phosphate linkages. In some embodiments, 65% or moreof the internucleotidic linkages of a wing region are natural phosphatelinkages. In some embodiments, 70% or more of the internucleotidiclinkages of a wing region are natural phosphate linkages. In someembodiments, 75% or more of the internucleotidic linkages of a wingregion are natural phosphate linkages. In some embodiments, 80% or moreof the internucleotidic linkages of a wing region are natural phosphatelinkages. In some embodiments, 85% or more of the internucleotidiclinkages of a wing region are natural phosphate linkages. In someembodiments, 90% or more of the internucleotidic linkages of a wingregion are natural phosphate linkages. In some embodiments, 95% or moreof the internucleotidic linkages of a wing region are natural phosphatelinkages. In some embodiments, each internucleotidic linkage of a wingregion is a natural phosphate linkage.

In some embodiments, a core region comprises 2 or more modifiedinternucleotidic linkages. In some embodiments, a core region comprises3 or more modified internucleotidic linkages. In some embodiments, acore region comprises 4 or more modified internucleotidic linkages. Insome embodiments, a core region comprises 5 or more modifiedinternucleotidic linkages. In some embodiments, a core region comprises6 or more modified internucleotidic linkages. In some embodiments, acore region comprises 7 or more modified internucleotidic linkages. Insome embodiments, a core region comprises 8 or more modifiedinternucleotidic linkages. In some embodiments, a core region comprises9 or more modified internucleotidic linkages. In some embodiments, acore region comprises 10 or more modified internucleotidic linkages. Insome embodiments, a core region comprises 11 or more modifiedinternucleotidic linkages. In some embodiments, a core region comprises12 or more modified internucleotidic linkages. In some embodiments, acore region comprises 13 or more modified internucleotidic linkages. Insome embodiments, a core region comprises 14 or more modifiedinternucleotidic linkages. In some embodiments, a core region comprises15 or more modified internucleotidic linkages. In some embodiments, acore region comprises 2 or consecutive modified internucleotidiclinkages. In some embodiments, a core region comprises 3 or consecutivemodified internucleotidic linkages. In some embodiments, a core regioncomprises 4 or consecutive modified internucleotidic linkages. In someembodiments, a core region comprises 5 or consecutive modifiedinternucleotidic linkages. In some embodiments, a core region comprises6 or consecutive modified internucleotidic linkages. In someembodiments, a core region comprises 7 or consecutive modifiedinternucleotidic linkages. In some embodiments, a core region comprises8 or consecutive modified internucleotidic linkages. In someembodiments, a core region comprises 9 or consecutive modifiedinternucleotidic linkages. In some embodiments, a core region comprises10 or consecutive modified internucleotidic linkages. In someembodiments, a core region comprises 11 or consecutive modifiedinternucleotidic linkages. In some embodiments, a core region comprises12 or consecutive modified internucleotidic linkages. In someembodiments, a core region comprises 13 or consecutive modifiedinternucleotidic linkages. In some embodiments, a core region comprises14 or consecutive modified internucleotidic linkages. In someembodiments, a core region comprises 15 or consecutive modifiedinternucleotidic linkages. In some embodiments, each internucleotidiclinkage in a core region is independently a modified internucleotidiclinkage.

In some embodiments, 5% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 10% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 15% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 20% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 25% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 30% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 35% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 40% or more of the internucleotidic linkages ofprovided oligonucleotides are modified internucleotidic linkages. Insome embodiments, 45% or more of the internucleotidic linkages of a coreregion are modified internucleotidic linkages. In some embodiments, 50%or more of the internucleotidic linkages of a core region are modifiedinternucleotidic linkages. In some embodiments, 55% or more of theinternucleotidic linkages of a core region are modified internucleotidiclinkages. In some embodiments, 60% or more of the internucleotidiclinkages of a core region are modified internucleotidic linkages. Insome embodiments, 65% or more of the internucleotidic linkages of a coreregion are modified internucleotidic linkages. In some embodiments, 70%or more of the internucleotidic linkages of a core region are modifiedinternucleotidic linkages. In some embodiments, 75% or more of theinternucleotidic linkages of a core region are modified internucleotidiclinkages. In some embodiments, 80% or more of the internucleotidiclinkages of a core region are modified internucleotidic linkages. Insome embodiments, 85% or more of the internucleotidic linkages of a coreregion are modified internucleotidic linkages. In some embodiments, 90%or more of the internucleotidic linkages of a core region are modifiedinternucleotidic linkages. In some embodiments, 95% or more of theinternucleotidic linkages of a core region are modified internucleotidiclinkages. In some embodiments, each internucleotidic linkage of a coreregion is a modified internucleotidic linkage.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising a first plurality ofoligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that apredetermined level of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

In some embodiments, a common base sequence and length may be referredto as a common base sequence. In some embodiments, oligonucleotideshaving a common base sequence may have the same pattern of nucleosidemodifications, e.g., sugar modifications, base modifications, etc. Insome embodiments, a pattern of nucleoside modifications may berepresented by a combination of locations and modifications. Forexample, for WV-1092, the pattern of nucleoside linkage is 5×2′-OMe(2′-OMe modification on sugar moieties)-DNA (no 2′-modifications on thesugar moiety)-5×2′-OMe from the 5′-end to the 3′-end. In someembodiments, a pattern of backbone linkages comprises locations andtypes (e.g., phosphate, phosphorothioate, substituted phosphorothioate,etc.) of each internucleotidic linkages. For example, for WV-1092, thepattern of backbone linkages is 1×PS(phosphorothioate)-3×PO(phosphate)-11×PS-3×PO-1×PS. A pattern of backbone chiral centers of anoligonucleotide can be designated by a combination of linkage phosphorusstereochemistry (Rp/Sp) from 5′ to 3′. For example, WV-1092 has apattern of 1S-3PO (phosphate)-8S-1R-2S-3PO-1S. In some embodiments, allnon-chiral linkages (e.g., PO) may be omitted. As exemplified above,locations of non-chiral linkages may be obtained, for example, frompattern of backbone linkages.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising a first plurality ofoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.

As understood by a person having ordinary skill in the art, astereorandom or racemic preparation of oligonucleotides is prepared bynon-stereoselective and/or low-stereoselective coupling of nucleotidemonomers, typically without using any chiral auxiliaries, chiralmodification reagents, and/or chiral catalysts. In some embodiments, ina substantially racemic (or chirally uncontrolled) preparation ofoligonucleotides, all or most coupling steps are not chirally controlledin that the coupling steps are not specifically conducted to provideenhanced stereoselectivity. An example substantially racemic preparationof oligonucleotides is the preparation of phosphorothioateoligonucleotides through sulfurizing phosphite triesters from commonlyused phosphoramidite oligonucleotide synthesis with eithertteraethylthiuram disulfide or (TETD) or 3H-1,2-bensodithiol-3-one1,1-dioxide (BDTD), a well-known process in the art. In someembodiments, substantially racemic preparation of oligonucleotidesprovides substantially racemic oligonucleotide compositions (or chirallyuncontrolled oligonucleotide compositions). In some embodiments, atleast one coupling of a nucleotide monomer has a diastereoselectivitylower than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3,98:2, or 99:1. In some embodiments, at least two couplings of anucleotide monomer have a diastereoselectivity lower than about 60:40,70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In someembodiments, at least three couplings of a nucleotide monomer have adiastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10,91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least fourcouplings of a nucleotide monomer have a diastereoselectivity lower thanabout 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or99:1. In some embodiments, at least five couplings of a nucleotidemonomer have a diastereoselectivity lower than about 60:40, 70:30,80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In someembodiments, in a stereorandom or racemic preparations, at least oneinternucleotidic linkage has a diastereoselectivity lower than about60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. Insome embodiments, at least two internucleotidic linkages have adiastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10,91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least threeinternucleotidic linkages have a diastereoselectivity lower than about60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. Insome embodiments, at least four internucleotidic linkages have adiastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10,91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at least fiveinternucleotidic linkages have a diastereoselectivity lower than about60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. Insome embodiments, a diastereoselectivity is lower than about 60:40. Insome embodiments, a diastereoselectivity is lower than about 70:30. Insome embodiments, a diastereoselectivity is lower than about 80:20. Insome embodiments, a diastereoselectivity is lower than about 90:10. Insome embodiments, a diastereoselectivity is lower than about 91:9. Insome embodiments, a diastereoselectivity is lower than about 92:8. Insome embodiments, a diastereoselectivity is lower than about 93:7. Insome embodiments, a diastereoselectivity is lower than about 94:6. Insome embodiments, a diastereoselectivity is lower than about 95:5. Insome embodiments, a diastereoselectivity is lower than about 96:4. Insome embodiments, a diastereoselectivity is lower than about 97:3. Insome embodiments, a diastereoselectivity is lower than about 98:2. Insome embodiments, a diastereoselectivity is lower than about 99:1. Insome embodiments, at least one coupling has a diastereoselectivity lowerthan about 90:10. In some embodiments, at least two couplings have adiastereoselectivity lower than about 90:10. In some embodiments, atleast three couplings have a diastereoselectivity lower than about90:10. In some embodiments, at least four couplings have adiastereoselectivity lower than about 90:10. In some embodiments, atleast five couplings have a diastereoselectivity lower than about 90:10.In some embodiments, at least one internucleotidic linkage has adiastereoselectivity lower than about 90:10. In some embodiments, atleast two internucleotidic linkages have a diastereoselectivity lowerthan about 90:10. In some embodiments, at least three internucleotidiclinkages have a diastereoselectivity lower than about 90:10. In someembodiments, at least four internucleotidic linkages have adiastereoselectivity lower than about 90:10. In some embodiments, atleast five internucleotidic linkages have a diastereoselectivity lowerthan about 90:10.

As understood by a person having ordinary skill in the art, in someembodiments, diastereoselectivity of a coupling or a linkage can beassessed through the diastereoselectivity of a dimer formation under thesame or comparable conditions, wherein the dimer has the same 5′- and3′-nucleosides and internucleotidic linkage. For example,diastereoselectivity of the underlined coupling or linkage in WV-1092mG*SmGmCmAmC*SA*SA*SG*SG*S G*SC*SA*SC*RA*SG*SmAmCmUmU*SmC (SEQ ID NO: 9)can be assessed from coupling two G moieties under the same orcomparable conditions, e.g., monomers, chiral auxiliaries, solvents,activators, temperatures, etc.

In some embodiments, the present disclosure provides chirally controlled(and/or stereochemically pure) oligonucleotide compositions comprising afirst plurality of oligonucleotides defined by having:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is asubstantially pure preparation of a single oligonucleotide in that atleast about 10% of the oligonucleotides in the composition have thecommon base sequence and length, the common pattern of backbonelinkages, and the common pattern of backbone chiral centers.

In some embodiments, the present disclosure provides chirally controlledoligonucleotide composition of a first plurality of oligonucleotides inthat the composition is enriched, relative to a substantially racemicpreparation of the same oligonucleotides, for oligonucleotides of asingle oligonucleotide type. In some embodiments, the present disclosureprovides chirally controlled oligonucleotide composition of a firstplurality of oligonucleotides in that the composition is enriched,relative to a substantially racemic preparation of the sameoligonucleotides, for oligonucleotides of a single oligonucleotide typethat share:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition comprising a first plurality ofoligonucleotides of a particular oligonucleotide type characterized by:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers;

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence and length, for oligonucleotides of theparticular oligonucleotide type.

In some embodiments, oligonucleotides having a common base sequence andlength, a common pattern of backbone linkages, and a common pattern ofbackbone chiral centers have a common pattern of backbone phosphorusmodifications and a common pattern of base modifications. In someembodiments, oligonucleotides having a common base sequence and length,a common pattern of backbone linkages, and a common pattern of backbonechiral centers have a common pattern of backbone phosphorusmodifications and a common pattern of nucleoside modifications. In someembodiments, oligonucleotides having a common base sequence and length,a common pattern of backbone linkages, and a common pattern of backbonechiral centers have identical structures.

In some embodiments, oligonucleotides of an oligonucleotide type have acommon pattern of backbone phosphorus modifications and a common patternof sugar modifications. In some embodiments, oligonucleotides of anoligonucleotide type have a common pattern of backbone phosphorusmodifications and a common pattern of base modifications. In someembodiments, oligonucleotides of an oligonucleotide type have a commonpattern of backbone phosphorus modifications and a common pattern ofnucleoside modifications. In some embodiments, oligonucleotides of anoligonucleotide type are identical.

In some embodiments, a chirally controlled oligonucleotide compositionis a substantially pure preparation of an oligonucleotide type in thatoligonucleotides in the composition that are not of the oligonucleotidetype are impurities form the preparation process of said oligonucleotidetype, in some case, after certain purification procedures.

In some embodiments, at least about 20% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 25% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 30% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 35% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 40% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 45% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 50% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 55% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 60% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 65% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 70% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 75% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 80% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 85% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 90% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 92% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 94% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 95% of the oligonucleotides in thecomposition have a common base sequence and length, a common pattern ofbackbone linkages, and a common pattern of backbone chiral centers. Insome embodiments, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% of the oligonucleotides in the composition have a common basesequence and length, a common pattern of backbone linkages, and a commonpattern of backbone chiral centers. In some embodiments, greater thanabout 99% of the oligonucleotides in the composition have a common basesequence and length, a common pattern of backbone linkages, and a commonpattern of backbone chiral centers. In some embodiments, purity of achirally controlled oligonucleotide composition of an oligonucleotidecan be expressed as the percentage of oligonucleotides in thecomposition that have a common base sequence and length, a commonpattern of backbone linkages, and a common pattern of backbone chiralcenters.

In some embodiments, oligonucleotides having a common base sequence andlength, a common pattern of backbone linkages, and a common pattern ofbackbone chiral centers have a common pattern of backbone phosphorusmodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of nucleosidemodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of sugarmodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of basemodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers have a common pattern ofbackbone phosphorus modifications and a common pattern of nucleosidemodifications. In some embodiments, oligonucleotides having a commonbase sequence and length, a common pattern of backbone linkages, and acommon pattern of backbone chiral centers are identical.

In some embodiments, oligonucleotides in provided compositions have acommon pattern of backbone phosphorus modifications. In someembodiments, a common base sequence is a base sequence of anoligonucleotide type. In some embodiments, a provided composition is anoligonucleotide composition that is chirally controlled in that thecomposition contains a predetermined level of a first plurality ofoligonucleotides of an individual oligonucleotide type, wherein anoligonucleotide type is defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

As noted above and understood in the art, in some embodiments, basesequence of an oligonucleotide may refer to the identity and/ormodification status of nucleoside residues (e.g., of sugar and/or basecomponents, relative to standard naturally occurring nucleotides such asadenine, cytosine, guanosine, thymine, and uracil) in theoligonucleotide and/or to the hybridization character (i.e., the abilityto hybridize with particular complementary residues) of such residues.

In some embodiments, a particular oligonucleotide type may be defined by

1A) base identity;

1B) pattern of base modification;

1C) pattern of sugar modification;

-   -   2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

Thus, in some embodiments, oligonucleotides of a particular type mayshare identical bases but differ in their pattern of base modificationsand/or sugar modifications. In some embodiments, oligonucleotides of aparticular type may share identical bases and pattern of basemodifications (including, e.g., absence of base modification), butdiffer in pattern of sugar modifications.

In some embodiments, oligonucleotides of a particular type are identicalin that they have the same base sequence (including length), the samepattern of chemical modifications to sugar and base moieties, the samepattern of backbone linkages (e.g., pattern of natural phosphatelinkages, phosphorothioate linkages, phosphorothioate triester linkages,and combinations thereof), the same pattern of backbone chiral centers(e.g., pattern of stereochemistry (Rp/Sp) of chiral internucleotidiclinkages), and the same pattern of backbone phosphorus modifications(e.g., pattern of modifications on the internucleotidic phosphorus atom,such as —S—, and -L-R¹ of formula I).

In some embodiments, purity of a chirally controlled oligonucleotidecomposition of an oligonucleotide type is expressed as the percentage ofoligonucleotides in the composition that are of the oligonucleotidetype. In some embodiments, at least about 10% of the oligonucleotides ina chirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 20% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 30% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 40% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 50% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 60% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 70% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 80% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 90% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 92% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 94% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 95% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 96% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type. In some embodiments, at least about 97% of theoligonucleotides in a chirally controlled oligonucleotide compositionare of the same oligonucleotide type. In some embodiments, at leastabout 98% of the oligonucleotides in a chirally controlledoligonucleotide composition are of the same oligonucleotide type. Insome embodiments, at least about 99% of the oligonucleotides in achirally controlled oligonucleotide composition are of the sameoligonucleotide type.

In some embodiments, purity of a chirally controlled oligonucleotidecomposition can be controlled by stereoselectivity of each coupling stepin its preparation process. In some embodiments, a coupling step has astereoselectivity (e.g., diastereoselectivity) of 60% (60% of the newinternucleotidic linkage formed from the coupling step has the intendedstereochemistry). After such a coupling step, the new internucleotidiclinkage formed may be referred to have a 60% purity. In someembodiments, each coupling step has a stereoselectivity of at least 60%.In some embodiments, each coupling step has a stereoselectivity of atleast 70%. In some embodiments, each coupling step has astereoselectivity of at least 80%. In some embodiments, each couplingstep has a stereoselectivity of at least 85%. In some embodiments, eachcoupling step has a stereoselectivity of at least 90%. In someembodiments, each coupling step has a stereoselectivity of at least 91%.In some embodiments, each coupling step has a stereoselectivity of atleast 92%. In some embodiments, each coupling step has astereoselectivity of at least 93%. In some embodiments, each couplingstep has a stereoselectivity of at least 94%. In some embodiments, eachcoupling step has a stereoselectivity of at least 95%. In someembodiments, each coupling step has a stereoselectivity of at least 96%.In some embodiments, each coupling step has a stereoselectivity of atleast 97%. In some embodiments, each coupling step has astereoselectivity of at least 98%. In some embodiments, each couplingstep has a stereoselectivity of at least 99%. In some embodiments, eachcoupling step has a stereoselectivity of at least 99.5%. In someembodiments, each coupling step has a stereoselectivity of virtually100%. In some embodiments, a coupling step has a stereoselectivity ofvirtually 100% in that all detectable product from the coupling step byan analytical method (e.g., NMR, HPLC, etc) has the intendedstereoselectivity.

Among other things, the present disclosure recognizes that combinationsof oligonucleotide structural elements (e.g., patterns of chemicalmodifications, backbone linkages, backbone chiral centers, and/orbackbone phosphorus modifications) can provide surprisingly improvedproperties such as bioactivities.

In some embodiments, the present disclosure provides an oligonucleotidecomposition comprising a predetermined level of a first plurality ofoligonucleotides which comprise one or more wing regions and a commoncore region, wherein:

each wing region independently has a length of two or more bases, andindependently and optionally comprises one or more chiralinternucleotidic linkages;

the core region independently has a length of two or more bases, andindependently comprises one or more chiral internucleotidic linkages,and the common core region has:

1) a common base sequence and length;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers.

In some embodiments, a wing region comprises a structural feature thatis not in a core region. In some embodiments, a wing and core can bedefined by any structural elements, e.g., base modifications (e.g.,methylated/non-methylated, methylation at position 1/methylation atposition 2, etc.), sugar modifications (e.g., modified/non-modified,2′-modification/another type of modification, one type of2′-modification/another type of 2′-modification, etc.), backbone linkagetypes (e.g., phosphate/phosphorothioate, phosphorothioate/substitutedphosphorothioate, etc.), backbone chiral center stereochemistry(e.g.,all Sp/all Rp, (SpRp) repeats/all Rp, etc.), backbone phosphorusmodification types (e.g., s1/s2, s1/s3, etc.), etc.

In some embodiments, a wing and core is defined by nucleosidemodifications, wherein a wing comprises a nucleoside modification thatthe core region does not have. In some embodiments, a wing and core isdefined by sugar modifications, wherein a wing comprises a sugarmodification that the core region does not have. In some embodiments, asugar modification is a 2′-modification. In some embodiments, a sugarmodification is 2′-OR¹. In some embodiments, a sugar modification is2′-MOE. In some embodiments, a sugar modification is 2′-OMe.Additionally example sugar modifications are described in the presentdisclosure. In some embodiments, a wing and core is defined byinternucleotidic linkages, wherein a wing comprises a internucleotidiclinkage type (e.g., natural phosphate linkage, a type of modifiedinternucleotidic linkage, etc.) that the core region does not have. Insome embodiments, a wing and core is defined by internucleotidiclinkages, wherein a wing has a pattern of backbone linkage that isdifferent from that of the core.

In some embodiments, oligonucleotides in provided compositions have awing-core structure (hemimer). In some embodiments, oligonucleotides inprovided compositions have a wing-core structure of nucleosidemodifications. In some embodiments, oligonucleotides in providedcompositions have a core-wing structure (another type of hemimer). Insome embodiments, oligonucleotides in provided compositions have acore-wing structure of nucleoside modifications. In some embodiments,oligonucleotides in provided compositions have a wing-core-wingstructure (gapmer). In some embodiments, oligonucleotides in providedcompositions have a wing-core-wing structure of nucleosidemodifications. In some embodiments, a wing and core is defined bymodifications of the sugar moieties. In some embodiments, a wing andcore is defined by modifications of the base moieties. In someembodiments, each sugar moiety in the wing region has the same2′-modification which is not found in the core region. In someembodiments, each sugar moiety in the wing region has the same2′-modification which is different than any sugar modifications in thecore region. In some embodiments, a core region has no sugarmodification. In some embodiments, each sugar moiety in the wing regionhas the same 2′-modification, and the core region has no2′-modifications. In some embodiments, when two or more wings arepresent, each wing is defined by its own modifications. In someembodiments, each wing has its own characteristic sugar modification. Insome embodiments, each wing has the same characteristic sugarmodification differentiating it from a core. In some embodiments, eachwing sugar moiety has the same modification. In some embodiments, eachwing sugar moiety has the same 2′-modification. In some embodiments,each sugar moiety in a wing region has the same 2′-modification, yet thecommon 2′-modification in a first wing region can either be the same asor different from the common 2′-modification in a second wing region. Insome embodiments, each sugar moiety in a wing region has the same2′-modification, and the common 2′-modification in a first wing regionis the same as the common 2′-modification in a second wing region. Insome embodiments, each sugar moiety in a wing region has the same2′-modification, and the common 2′-modification in a first wing regionis different from the common 2′-modification in a second wing region.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are antisense oligonucleotides(e.g., chiromersen). In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are siRNA oligonucleotides.In some embodiments, a provided chirally controlled oligonucleotidecomposition is of oligonucleotides that can be antisenseoligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir,ribozyme, U1 adaptor, RNA activator, RNAi agent, decoy oligonucleotide,triplex forming oligonucleotide, aptamer or adjuvant. In someembodiments, a chirally controlled oligonucleotide composition is ofantisense oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of antagomir oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofmicroRNA oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of pre-microRNA oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofantimir oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of supermir oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofribozyme oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of U1 adaptor oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is of RNAactivator oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of RNAi agent oligonucleotides. In someembodiments, a chirally controlled oligonucleotide composition is ofdecoy oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of triplex forming oligonucleotides. Insome embodiments, a chirally controlled oligonucleotide composition isof aptamer oligonucleotides. In some embodiments, a chirally controlledoligonucleotide composition is of adjuvant oligonucleotides.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides that includeone or more modified backbone linkages, bases, and/or sugars.

In some embodiments, a provided oligonucleotide comprises one or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide comprises two or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide comprisesthree or more chiral, modified phosphate linkages. In some embodiments,a provided oligonucleotide comprises four or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotidecomprises five or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25chiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 5 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises6 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 7 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 8 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 9 or more chiral,modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 10 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises11 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 12 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 13 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 14 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 15 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises16 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 17 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 18 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 19 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 20 or more chiral, modified phosphatelinkages. In some embodiments, a provided oligonucleotide type comprises21 or more chiral, modified phosphate linkages. In some embodiments, aprovided oligonucleotide type comprises 22 or more chiral, modifiedphosphate linkages. In some embodiments, a provided oligonucleotide typecomprises 23 or more chiral, modified phosphate linkages. In someembodiments, a provided oligonucleotide type comprises 24 or morechiral, modified phosphate linkages. In some embodiments, a providedoligonucleotide type comprises 25 or more chiral, modified phosphatelinkages.

In some embodiments, a provided oligonucleotide comprises at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% chiral, modified phosphate linkages. Examplesuch chiral, modified phosphate linkages are described above and herein.In some embodiments, a provided oligonucleotide comprises at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% chiral, modified phosphate linkages in theSp configuration.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 80%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 85%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 90%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 91%.In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 92%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 93%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 94%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 95%.In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of a stereochemical purity ofgreater than about 96%. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are of astereochemical purity of greater than about 97%. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of a stereochemical purity of greater than about 98%. In someembodiments, provided chirally controlled (and/or stereochemically pure)preparations are of a stereochemical purity of greater than about 99%.

In some embodiments, a chiral, modified phosphate linkage is a chiralphosphorothioate linkage, i.e., phosphorothioate internucleotidiclinkage. In some embodiments, a provided oligonucleotide comprises atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral phosphorothioateinternucleotidic linkages. In some embodiments, all chiral, modifiedphosphate linkages are chiral phosphorothioate internucleotidiclinkages. In some embodiments, at least about 10, 20, 30, 40, 50, 60,70, 80, or 90% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 10% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Sp conformation. Insome embodiments, at least about 20% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Spconformation. In some embodiments, at least about 30% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Sp conformation. In some embodiments, at least about 40%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Sp conformation. In some embodiments, atleast about 50% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation. In someembodiments, at least about 60% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Sp conformation. Insome embodiments, at least about 70% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Spconformation. In some embodiments, at least about 80% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Sp conformation. In some embodiments, at least about 90%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Sp conformation. In some embodiments, atleast about 95% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Sp conformation.

In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or90% chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, atleast about 10% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, at least about 20% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Rp conformation. Insome embodiments, at least about 30% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, at least about 40% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, at least about 50%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, atleast about 60% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, at least about 70% chiral phosphorothioate internucleotidiclinkages of a provided oligonucleotide are of the Rp conformation. Insome embodiments, at least about 80% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, at least about 90% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, at least about 95%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation.

In some embodiments, less than about 10, 20, 30, 40, 50, 60, 70, 80, or90% chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 10% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, less than about 20% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, less than about 30% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 40%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 50% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, less than about 60% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, less than about 70% chiralphosphorothioate internucleotidic linkages of a provided oligonucleotideare of the Rp conformation. In some embodiments, less than about 80%chiral phosphorothioate internucleotidic linkages of a providedoligonucleotide are of the Rp conformation. In some embodiments, lessthan about 90% chiral phosphorothioate internucleotidic linkages of aprovided oligonucleotide are of the Rp conformation. In someembodiments, less than about 95% chiral phosphorothioateinternucleotidic linkages of a provided oligonucleotide are of the Rpconformation. In some embodiments, a provided oligonucleotide has onlyone Rp chiral phosphorothioate internucleotidic linkages. In someembodiments, a provided oligonucleotide has only one Rp chiralphosphorothioate internucleotidic linkages, wherein all internucleotidelinkages are chiral phosphorothioate internucleotidic linkages.

In some embodiments, a chiral phosphorothioate internucleotidic linkageis a chiral phosphorothioate diester linkage. In some embodiments, eachchiral phosphorothioate internucleotidic linkage is independently achiral phosphorothioate diester linkage. In some embodiments, eachinternucleotidic linkage is independently a chiral phosphorothioatediester linkage. In some embodiments, each internucleotidic linkage isindependently a chiral phosphorothioate diester linkage, and only one isRp.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides that containone or more modified bases. In some embodiments, provided chirallycontrolled (and/or stereochemically pure) preparations are ofoligonucleotides that contain no modified bases. Example such modifiedbases are described above and herein.

In some embodiments, oligonucleotides of provided compositions compriseat least 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. Insome embodiments, oligonucleotides of provided compositions comprise atleast one natural phosphate linkage. In some embodiments,oligonucleotides of provided compositions comprise at least two naturalphosphate linkages. In some embodiments, oligonucleotides of providedcompositions comprise at least three natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise at leastfour natural phosphate linkages. In some embodiments, oligonucleotidesof provided compositions comprise at least five natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise at least six natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least sevennatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise at least eight natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise at least nine natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least ten naturalphosphate linkages.

In some embodiments, oligonucleotides of provided compositions comprise2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise onenatural phosphate linkage. In some embodiments, oligonucleotides ofprovided compositions comprise two natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise threenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise four natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise fivenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise six natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise sevennatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise eight natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise ninenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise ten natural phosphate linkages.

In some embodiments, oligonucleotides of provided compositions compriseat least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise at least two consecutive natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise at leastthree consecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least fourconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least fiveconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least sixconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least sevenconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least eightconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least nineconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise at least tenconsecutive natural phosphate linkages.

In some embodiments, oligonucleotides of provided compositions comprise2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive natural phosphate linkages. Insome embodiments, oligonucleotides of provided compositions comprise twoconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise three consecutivenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise four consecutive natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise five consecutive natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise sixconsecutive natural phosphate linkages. In some embodiments,oligonucleotides of provided compositions comprise seven consecutivenatural phosphate linkages. In some embodiments, oligonucleotides ofprovided compositions comprise eight consecutive natural phosphatelinkages. In some embodiments, oligonucleotides of provided compositionscomprise nine consecutive natural phosphate linkages. In someembodiments, oligonucleotides of provided compositions comprise tenconsecutive natural phosphate linkages.

In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 8 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 9 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 10 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 11 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 12bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 13 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 14 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 15 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 16 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 17bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 18 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 19 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 20 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 21 bases. In some embodiments,provided chirally controlled (and/or stereochemically pure) preparationsare of oligonucleotides having a common base sequence of at least 22bases. In some embodiments, provided chirally controlled (and/orstereochemically pure) preparations are of oligonucleotides having acommon base sequence of at least 23 bases. In some embodiments, providedchirally controlled (and/or stereochemically pure) preparations are ofoligonucleotides having a common base sequence of at least 24 bases. Insome embodiments, provided chirally controlled (and/or stereochemicallypure) preparations are of oligonucleotides having a common base sequenceof at least 25 bases. In some embodiments, provided chirally controlled(and/or stereochemically pure) preparations are of oligonucleotideshaving a common base sequence of at least 30, 35, 40, 45, 50, 55, 60,65, 70, or 75 bases.

In some embodiments, provided compositions comprise oligonucleotidescontaining one or more residues which are modified at the sugar moiety.In some embodiments, provided compositions comprise oligonucleotidescontaining one or more residues which are modified at the 2′ position ofthe sugar moiety (referred to herein as a “2′-modification”). Examplesuch modifications are described above and herein and include, but arenot limited to, 2′-OMe, 2′-MOE, 2′-LNA, 2′-F, FRNA, FANA, S-cEt, etc. Insome embodiments, provided compositions comprise oligonucleotidescontaining one or more residues which are 2′-modified. For example, insome embodiments, provided oligonucleotides contain one or more residueswhich are 2′-O-methoxyethyl (2′-MOE)-modified residues. In someembodiments, provided compositions comprise oligonucleotides which donot contain any 2′-modifications. In some embodiments, providedcompositions are oligonucleotides which do not contain any 2′-MOEresidues. That is, in some embodiments, provided oligonucleotides arenot MOE-modified. Additional example sugar modifications are describedin the present disclosure.

In some embodiments, provided oligonucleotides are of a general motif ofwing-core or core-wing (hemimer, also represented herein generally asX—Y or Y—X, respectively). In some embodiments, providedoligonucleotides are of a general motif of wing-core-wing (gapmer, alsorepresented herein generally as X—Y—X). In some embodiments, each wingregion independently contains one or more residues having a particularmodification, which modification is absent from the core “Y” portion. Insome embodiments, each wing region independently contains one or moreresidues having a particular nucleoside modification, which modificationis absent from the core “Y” portion. In some embodiments, each wingregion independently contains one or more residues having a particularbase modification, which modification is absent from the core “Y”portion. In some embodiments, each wing region independently containsone or more residues having a particular sugar modification, whichmodification is absent from the core “Y” portion. Example sugarmodifications are widely known in the art. In some embodiments, a sugarmodification is a modification selected from those modificationsdescribed in U.S. Pat. No. 9,006,198, which sugar modifications areincorporated herein by references. Additional example sugarmodifications are described in the present disclosure. In someembodiment, each wing contains one or more residues having a 2′modification that is not present in the core portion. In someembodiments, a 2′-modification is 2′-OR¹, wherein R¹ is as defined anddescribed in the present disclosure.

In some embodiments, provided oligonucleotides have a wing-core motifrepresented as X—Y, or a core-wing motif represented as Y—X, wherein theresidues at the “X” portion are sugar modified residues of a particulartype and the residues in the core “Y” portion are not sugar modifiedresidues of the same particular type. In some embodiments, providedoligonucleotides have a wing-core-wing motif represented as X—Y—X,wherein the residues at each “X” portion are sugar modified residues ofa particular type and the residues in the core “Y” portion are not sugarmodified residues of the same particular type. In some embodiments,provided oligonucleotides have a wing-core motif represented as X—Y, ora core-wing motif represented as Y—X, wherein the residues at the “X”portion are 2′-modified residues of a particular type and the residuesin the core “Y” portion are not 2′-modified residues of the sameparticular type. In some embodiments, provided oligonucleotides have awing-core motif represented as X—Y, wherein the residues at the “X”portion are 2′-modified residues of a particular type and the residuesin the core “Y” portion are not 2′-modified residues of the sameparticular type. In some embodiments, provided oligonucleotides have acore-wing motif represented as Y—X, wherein the residues at the “X”portion are 2′-modified residues of a particular type and the residuesin the core “Y” portion are not 2′-modified residues of the sameparticular type. In some embodiments, provided oligonucleotides have awing-core-wing motif represented as X—Y—X, wherein the residues at each“X” portion are 2′-modified residues of a particular type and theresidues in the core “Y” portion are not 2′-modified residues of thesame particular type. In some embodiments, provided oligonucleotideshave a wing-core motif represented as X—Y, wherein the residues at the“X” portion are 2′-modified residues of a particular type and theresidues in the core “Y” portion are 2′-deoxyribonucleoside. In someembodiments, provided oligonucleotides have a core-wing motifrepresented as Y—X, wherein the residues at the “X” portion are2′-modified residues of a particular type and the residues in the core“Y” portion are 2′-deoxyribonucleoside. In some embodiments, providedoligonucleotides have a wing-core-wing motif represented as X—Y—X,wherein the residues at each “X” portion are 2′-modified residues of aparticular type and the residues in the core “Y” portion are2′-deoxyribonucleoside. In some embodiments, provided oligonucleotideshave a wing-core-wing motif represented as X—Y—X, wherein the residuesat each “X” portion are 2′-modified residues of a particular type andthe residues in the core “Y” portion are 2′-deoxyribonucleoside. Forinstance, in some embodiments, provided oligonucleotides have awing-core-wing motif represented as X—Y—X, wherein the residues at each“X” portion are 2′-MOE-modified residues and the residues in the core“Y” portion are not 2′-MOE-modified residues. In some embodiments,provided oligonucleotides have a wing-core-wing motif represented asX—Y—X, wherein the residues at each “X” portion are 2′-MOE-modifiedresidues and the residues in the core “Y” portion are2′-deoxyribonucleoside. One of skill in the relevant arts will recognizethat all such 2′-modifications described above and herein arecontemplated in the context of such X—Y, Y—X and/or X—Y—X motifs.

In some embodiments, a wing has a length of one or more bases. In someembodiments, a wing has a length of two or more bases. In someembodiments, a wing has a length of three or more bases. In someembodiments, a wing has a length of four or more bases. In someembodiments, a wing has a length of five or more bases. In someembodiments, a wing has a length of six or more bases. In someembodiments, a wing has a length of seven or more bases. In someembodiments, a wing has a length of eight or more bases. In someembodiments, a wing has a length of nine or more bases. In someembodiments, a wing has a length of ten or more bases. In someembodiments, a wing has a length of 11 or more bases. In someembodiments, a wing has a length of 12 or more bases. In someembodiments, a wing has a length of 13 or more bases. In someembodiments, a wing has a length of 14 or more bases. In someembodiments, a wing has a length of 15 or more bases. In someembodiments, a wing has a length of 16 or more bases. In someembodiments, a wing has a length of 17 or more bases. In someembodiments, a wing has a length of 18 or more bases. In someembodiments, a wing has a length of 19 or more bases. In someembodiments, a wing has a length often or more bases.

In some embodiments, a wing has a length of one base. In someembodiments, a wing has a length of two bases. In some embodiments, awing has a length of three bases. In some embodiments, a wing has alength of four bases. In some embodiments, a wing has a length of fivebases. In some embodiments, a wing has a length of six bases. In someembodiments, a wing has a length of seven bases. In some embodiments, awing has a length of eight bases. In some embodiments, a wing has alength of nine bases. In some embodiments, a wing has a length of tenbases. In some embodiments, a wing has a length of 11 bases. In someembodiments, a wing has a length of 12 bases. In some embodiments, awing has a length of 13 bases. In some embodiments, a wing has a lengthof 14 bases. In some embodiments, a wing has a length of 15 bases. Insome embodiments, a wing has a length of 16 bases. In some embodiments,a wing has a length of 17 bases. In some embodiments, a wing has alength of 18 bases. In some embodiments, a wing has a length of 19bases. In some embodiments, a wing has a length of ten bases.

In some embodiments, a wing comprises one or more chiralinternucleotidic linkages. In some embodiments, a wing comprises one ormore natural phosphate linkages. In some embodiments, a wing comprisesone or more chiral internucleotidic linkages and one or more naturalphosphate linkages. In some embodiments, a wing comprises one or morechiral internucleotidic linkages and two or more natural phosphatelinkages. In some embodiments, a wing comprises one or more chiralinternucleotidic linkages and two or more natural phosphate linkages,wherein two or more natural phosphate linkages are consecutive. In someembodiments, a wing comprises no chiral internucleotidic linkages. Insome embodiments, each wing linkage is a natural phosphate linkage. Insome embodiments, a wing comprises no phosphate linkages. In someembodiments, each wing is independently a chiral internucleotidiclinkage.

In some embodiments, each wing region independently comprises one ormore chiral internucleotidic linkages. In some embodiments, each wingregion independently comprises one or more natural phosphate linkages.In some embodiments, each wing region independently comprises one ormore chiral internucleotidic linkages and one or more natural phosphatelinkages. In some embodiments, each wing region independently comprisesone or more chiral internucleotidic linkages and two or more naturalphosphate linkages. In some embodiments, each wing region independentlycomprises one or more chiral internucleotidic linkages and two or morenatural phosphate linkages, wherein two or more natural phosphatelinkages are consecutive.

In some embodiments, each wing region independently comprises at leastone chiral internucleotidic linkage. In some embodiments, each wingregion independently comprises at least two chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least three chiral internucleotidic linkages. In some embodiments,each wing region independently comprises at least four chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least five chiral internucleotidic linkages.In some embodiments, each wing region independently comprises at leastsix chiral internucleotidic linkages. In some embodiments, each wingregion independently comprises at least seven chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least eight chiral internucleotidic linkages. In some embodiments,each wing region independently comprises at least nine chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least ten chiral internucleotidic linkages.In some embodiments, each wing region independently comprises at least11 chiral internucleotidic linkages. In some embodiments, each wingregion independently comprises at least 12 chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least 13 chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises at least 14 chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least 15 chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises at least 16 chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least 17 chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises at least 18 chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least 19 chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises at least 20 chiral internucleotidiclinkages.

In some embodiments, each wing region independently comprises one chiralinternucleotidic linkage. In some embodiments, each wing regionindependently comprises two chiral internucleotidic linkages. In someembodiments, each wing region independently comprises three chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises four chiral internucleotidic linkages. In someembodiments, each wing region independently comprises five chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises six chiral internucleotidic linkages. In someembodiments, each wing region independently comprises seven chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises eight chiral internucleotidic linkages. In someembodiments, each wing region independently comprises nine chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises ten chiral internucleotidic linkages. In someembodiments, each wing region independently comprises 11 chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 12 chiral internucleotidic linkages. In someembodiments, each wing region independently comprises 13 chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 14 chiral internucleotidic linkages. In someembodiments, each wing region independently comprises 15 chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 16 chiral internucleotidic linkages. In someembodiments, each wing region independently comprises 17 chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 18 chiral internucleotidic linkages. In someembodiments, each wing region independently comprises 19 chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 20 chiral internucleotidic linkages.

In some embodiments, each wing region independently comprises at leastone consecutive natural phosphate linkage. In some embodiments, eachwing region independently comprises at least two consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least three consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least four consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least five consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least six consecutive chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least seven consecutive chiral internucleotidic linkages. In someembodiments, each wing region independently comprises at least eightconsecutive chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises at least nine consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least ten consecutive chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least 11 consecutive chiral internucleotidic linkages. In someembodiments, each wing region independently comprises at least 12consecutive chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises at least 13 consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least 14 consecutive chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least 15 consecutive chiral internucleotidic linkages. In someembodiments, each wing region independently comprises at least 16consecutive chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises at least 17 consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises at least 18 consecutive chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesat least 19 consecutive chiral internucleotidic linkages. In someembodiments, each wing region independently comprises at least 20consecutive chiral internucleotidic linkages.

In some embodiments, each wing region independently comprises oneconsecutive natural phosphate linkage. In some embodiments, each wingregion independently comprises two consecutive chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesthree consecutive chiral internucleotidic linkages. In some embodiments,each wing region independently comprises four consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises five consecutive chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisessix consecutive chiral internucleotidic linkages. In some embodiments,each wing region independently comprises seven consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises eight consecutive chiral internucleotidiclinkages. In some embodiments, each wing region independently comprisesnine consecutive chiral internucleotidic linkages. In some embodiments,each wing region independently comprises ten consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 11 consecutive chiral internucleotidic linkages.In some embodiments, each wing region independently comprises 12consecutive chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises 13 consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 14 consecutive chiral internucleotidic linkages.In some embodiments, each wing region independently comprises 15consecutive chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises 16 consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 17 consecutive chiral internucleotidic linkages.In some embodiments, each wing region independently comprises 18consecutive chiral internucleotidic linkages. In some embodiments, eachwing region independently comprises 19 consecutive chiralinternucleotidic linkages. In some embodiments, each wing regionindependently comprises 20 consecutive chiral internucleotidic linkages.

In some embodiments, each wing region independently comprises at leastone natural phosphate linkage. In some embodiments, each wing regionindependently comprises at least two natural phosphate linkages. In someembodiments, each wing region independently comprises at least threenatural phosphate linkages. In some embodiments, each wing regionindependently comprises at least four natural phosphate linkages. Insome embodiments, each wing region independently comprises at least fivenatural phosphate linkages. In some embodiments, each wing regionindependently comprises at least six natural phosphate linkages. In someembodiments, each wing region independently comprises at least sevennatural phosphate linkages. In some embodiments, each wing regionindependently comprises at least eight natural phosphate linkages. Insome embodiments, each wing region independently comprises at least ninenatural phosphate linkages. In some embodiments, each wing regionindependently comprises at least ten natural phosphate linkages. In someembodiments, each wing region independently comprises at least 11natural phosphate linkages. In some embodiments, each wing regionindependently comprises at least 12 natural phosphate linkages. In someembodiments, each wing region independently comprises at least 13natural phosphate linkages. In some embodiments, each wing regionindependently comprises at least 14 natural phosphate linkages. In someembodiments, each wing region independently comprises at least 15natural phosphate linkages. In some embodiments, each wing regionindependently comprises at least 16 natural phosphate linkages. In someembodiments, each wing region independently comprises at least 17natural phosphate linkages. In some embodiments, each wing regionindependently comprises at least 18 natural phosphate linkages. In someembodiments, each wing region independently comprises at least 19natural phosphate linkages. In some embodiments, each wing regionindependently comprises at least 20 natural phosphate linkages.

In some embodiments, each wing region independently comprises onenatural phosphate linkage. In some embodiments, each wing regionindependently comprises two natural phosphate linkages. In someembodiments, each wing region independently comprises three naturalphosphate linkages. In some embodiments, each wing region independentlycomprises four natural phosphate linkages. In some embodiments, eachwing region independently comprises five natural phosphate linkages. Insome embodiments, each wing region independently comprises six naturalphosphate linkages. In some embodiments, each wing region independentlycomprises seven natural phosphate linkages. In some embodiments, eachwing region independently comprises eight natural phosphate linkages. Insome embodiments, each wing region independently comprises nine naturalphosphate linkages. In some embodiments, each wing region independentlycomprises ten natural phosphate linkages. In some embodiments, each wingregion independently comprises 11 natural phosphate linkages. In someembodiments, each wing region independently comprises 12 naturalphosphate linkages. In some embodiments, each wing region independentlycomprises 13 natural phosphate linkages. In some embodiments, each wingregion independently comprises 14 natural phosphate linkages. In someembodiments, each wing region independently comprises 15 naturalphosphate linkages. In some embodiments, each wing region independentlycomprises 16 natural phosphate linkages. In some embodiments, each wingregion independently comprises 17 natural phosphate linkages. In someembodiments, each wing region independently comprises 18 naturalphosphate linkages. In some embodiments, each wing region independentlycomprises 19 natural phosphate linkages. In some embodiments, each wingregion independently comprises 20 natural phosphate linkages.

In some embodiments, each wing region independently comprises at leastone consecutive natural phosphate linkage. In some embodiments, eachwing region independently comprises at least two consecutive naturalphosphate linkages. In some embodiments, each wing region independentlycomprises at least three consecutive natural phosphate linkages. In someembodiments, each wing region independently comprises at least fourconsecutive natural phosphate linkages. In some embodiments, each wingregion independently comprises at least five consecutive naturalphosphate linkages. In some embodiments, each wing region independentlycomprises at least six consecutive natural phosphate linkages. In someembodiments, each wing region independently comprises at least sevenconsecutive natural phosphate linkages. In some embodiments, each wingregion independently comprises at least eight consecutive naturalphosphate linkages. In some embodiments, each wing region independentlycomprises at least nine consecutive natural phosphate linkages. In someembodiments, each wing region independently comprises at least tenconsecutive natural phosphate linkages. In some embodiments, each wingregion independently comprises at least 11 consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprisesat least 12 consecutive natural phosphate linkages. In some embodiments,each wing region independently comprises at least 13 consecutive naturalphosphate linkages. In some embodiments, each wing region independentlycomprises at least 14 consecutive natural phosphate linkages. In someembodiments, each wing region independently comprises at least 15consecutive natural phosphate linkages. In some embodiments, each wingregion independently comprises at least 16 consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprisesat least 17 consecutive natural phosphate linkages. In some embodiments,each wing region independently comprises at least 18 consecutive naturalphosphate linkages. In some embodiments, each wing region independentlycomprises at least 19 consecutive natural phosphate linkages. In someembodiments, each wing region independently comprises at least 20consecutive natural phosphate linkages.

In some embodiments, each wing region independently comprises oneconsecutive natural phosphate linkage. In some embodiments, each wingregion independently comprises two consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprisesthree consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises four consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprisesfive consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises six consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprisesseven consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises eight consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprisesnine consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises ten consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprises11 consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises 12 consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprises13 consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises 14 consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprises15 consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises 16 consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprises17 consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises 18 consecutive natural phosphatelinkages. In some embodiments, each wing region independently comprises19 consecutive natural phosphate linkages. In some embodiments, eachwing region independently comprises 20 consecutive natural phosphatelinkages.

In some embodiments, a wing is to the 5′-end of a core (5′-end wing). Insome embodiments, a wing is to the 3′-end of a core (3′-end wing). Forexample, in WV-1092 (mG* SmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmU*SmC (SEQ ID NO: 10)), mG*SmGmCmAmC is a 5′-end wing,*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*S (SEQ ID NO: 11) is a core, andmAmCmUmU*SmC is a 3′-end wing.

In some embodiments, a 5′-end wing comprises one or more modifiedinternucleotidic linkages and one or more natural phosphateinternucleotidic linkages. In some embodiments, a 3′-end wing comprisesone or more modified internucleotidic linkages and one or more naturalphosphate internucleotidic linkages. In some embodiments, each wingindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate internucleotidic linkages. Forexample, WV-1092 has a 5′-end wing comprises one or more modifiedinternucleotidic linkages and one or more natural phosphateinternucleotidic linkages, and a 3′-end wing comprises one or moremodified internucleotidic linkages and one or more natural phosphateinternucleotidic linkages.

In some embodiments, a 5′-end wing comprises a modified internucleotidiclinkage having one or more natural phosphate linkages connecting two ormore nucleosides after (to the 3′-end) the modified internucleotidiclinkage in the 5′-end wing. For example, a 5′-end wing mG*SmGmCmAmCcomprises a modified internucleotidic linkage (mGSmG) which has threenatural phosphate linkages connecting four nucleosides (mGmCmAmC) afterthe modified internucleotidic linkage in the 5′-end wing. In someembodiments, a 5′-end wing comprises a modified internucleotidiclinkages followed by one or more natural phosphate linkages and/or oneor more modified internucleotidic linkages, which are followed by one ormore natural phosphate linkages in the 5′-end wing (for example, mG*SmGand mG*SmC in mG*SmG*SmCmAmC). In some embodiments, a 5′-end wingcomprises a modified internucleotidic linkages followed by one or morenatural phosphate linkages in the 5′-end wing. In some embodiments, a5′-end wing comprises a modified internucleotidic linkages followed byone or more consecutive natural phosphate linkages in the 5′-end wing.In some embodiments, a 5′-end wing comprises a natural phosphate linkagebetween the two nucleosides at its 3′-end. For example, a 5′-end wingmG*SmGmCmAmC has a natural phosphate linkage between the two nucleosidesat its 3′-end (mG*SmGmCmAmC).

In some embodiments, a 3′-end wing comprises a modified internucleotidiclinkage having one or more natural phosphate linkages connecting two ormore nucleosides before (to the 5′-end) the modified internucleotidiclinkage in the 3′-end wing. For example, a 3′-end wing mAmCmUmU*SmCcomprises a modified internucleotidic linkage (mU*SmC) which has threenatural phosphate linkages connecting four nucleosides (mAmCmUmU) beforethe modified internucleotidic linkage in the 3′-end wing. In someembodiments, a 3′-end wing comprises a modified internucleotidiclinkages preceded by one or more natural phosphate linkages and/or oneor more modified internucleotidic linkages, which are preceded by one ormore natural phosphate linkages in the 3′-end wing (for example, mU*SmUand mU*SmC in mAmCmU*SmU*SmC). In some embodiments, a 3′-end wingcomprises a modified internucleotidic linkages preceded by one or morenatural phosphate linkages in the 3′-end wing. In some embodiments, a3′-end wing comprises a modified internucleotidic linkages preceded byone or more consecutive natural phosphate linkages in the 3′-end wing.In some embodiments, a 3′-end wing comprises a natural phosphate linkagebetween the two nucleosides at its 5′-end. For example, a 3′-end winghaving the structure of mAmCmUmU*SmC has a natural phosphate linkagebetween the two nucleosides at its 5′-end (mAmCmUmU*SmC).

In some embodiments, one or more is one. In some embodiments, one ormore is two. In some embodiments, one or more is three. In someembodiments, one or more is four. In some embodiments, one or more isfive. In some embodiments, one or more is six. In some embodiments, oneor more is seven. In some embodiments, one or more is eight. In someembodiments, one or more is nine. In some embodiments, one or more isten. In some embodiments, one or more is at least one. In someembodiments, one or more is at least two. In some embodiments, one ormore is at least three. In some embodiments, one or more is at leastfour. In some embodiments, one or more is at least five. In someembodiments, one or more is at least six. In some embodiments, one ormore is at least seven. In some embodiments, one or more is at leasteight. In some embodiments, one or more is at least nine. In someembodiments, one or more is at least ten.

In some embodiments, a wing comprises only one chiral internucleotidiclinkage. In some embodiments, a 5′-end wing comprises only one chiralinternucleotidic linkage. In some embodiments, a 5′-end wing comprisesonly one chiral internucleotidic linkage at the 5′-end of the wing. Insome embodiments, a 5′-end wing comprises only one chiralinternucleotidic linkage at the 5′-end of the wing, and the chiralinternucleotidic linkage is Rp. In some embodiments, a 5′-end wingcomprises only one chiral internucleotidic linkage at the 5′-end of thewing, and the chiral internucleotidic linkage is Sp. In someembodiments, a 3′-end wing comprises only one chiral internucleotidiclinkage at the 3′-end of the wing. In some embodiments, a 3′-end wingcomprises only one chiral internucleotidic linkage at the 3′-end of thewing, and the chiral internucleotidic linkage is Rp. In someembodiments, a 3′-end wing comprises only one chiral internucleotidiclinkage at the 3′-end of the wing, and the chiral internucleotidiclinkage is Sp.

In some embodiments, a wing comprises two or more natural phosphatelinkages. In some embodiments, all phosphate linkages within a wing areconsecutive, and there are no non-phosphate linkages between any twophosphate linkages within a wing.

In some embodiments, a linkage connecting a wing and a core isconsidered part of the core when describing linkages, e.g., linkagechemistry, linkage stereochemistry, etc. For example, in WV-1092,mG*SmGmCmAmC*SA*SA*SG*SG*SG*SC*S A*SC*RA*SG*SmAmCmUmU*SmC (SEQ ID NO:12), the underlined linkages may be considered as part of the core(bold), its 5′-wing (having 2′-OMe on sugar moieties) has one single Spphosphorothioate linkages at its 5′-end, its 3′-wing (having 2′-OMe onsugar moieties) has one Sp phosphorothioate linkage at its 3′-end, andits core has no 2′-modifications on sugar).

In some embodiments, a 5′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a modified linkage. In someembodiments, a 5′-internucleotidic linkage connected to a sugar moietywithout a 2′-modification is a linkage having the structure of formulaI. In some embodiments, a 5′-internucleotidic linkage connected to asugar moiety without a 2′-modification is phosphorothioate linkage. Insome embodiments, a 5′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a substituted phosphorothioatelinkage. In some embodiments, a 5′-internucleotidic linkage connected toa sugar moiety without a 2′-modification is a phosphorothioate triesterlinkage. In some embodiments, each 5′-internucleotidic linkage connectedto a sugar moiety without a 2′-modification is a modified linkage. Insome embodiments, each 5′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a linkage having the structure offormula I. In some embodiments, each 5′-internucleotidic linkageconnected to a sugar moiety without a 2′-modification isphosphorothioate linkage. In some embodiments, each 5′-internucleotidiclinkage connected to a sugar moiety without a 2′-modification is asubstituted phosphorothioate linkage. In some embodiments, each5′-internucleotidic linkage connected to a sugar moiety without a2′-modification is a phosphorothioate triester linkage.

In some embodiments, a 3′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a modified linkage. In someembodiments, a 3′-internucleotidic linkage connected to a sugar moietywithout a 2′-modification is a linkage having the structure of formulaI. In some embodiments, a 3′-internucleotidic linkage connected to asugar moiety without a 2′-modification is phosphorothioate linkage. Insome embodiments, a 3′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a substituted phosphorothioatelinkage. In some embodiments, a 3′-internucleotidic linkage connected toa sugar moiety without a 2′-modification is a phosphorothioate triesterlinkage. In some embodiments, each 3′-internucleotidic linkage connectedto a sugar moiety without a 2′-modification is a modified linkage. Insome embodiments, each 3′-internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a linkage having the structure offormula I. In some embodiments, each 3′-internucleotidic linkageconnected to a sugar moiety without a 2′-modification isphosphorothioate linkage. In some embodiments, each 3′-internucleotidiclinkage connected to a sugar moiety without a 2′-modification is asubstituted phosphorothioate linkage. In some embodiments, each3′-internucleotidic linkage connected to a sugar moiety without a2′-modification is a phosphorothioate triester linkage.

In some embodiments, both internucleotidic linkages connected to a sugarmoiety without a 2′-modification are modified linkages. In someembodiments, both internucleotidic linkages connected to a sugar moietywithout a 2′-modification are linkage having the structure of formula I.In some embodiments, both internucleotidic linkages connected to a sugarmoiety without a 2′-modification are phosphorothioate linkages. In someembodiments, both internucleotidic linkages connected to a sugar moietywithout a 2′-modification are substituted phosphorothioate linkages. Insome embodiments, both internucleotidic linkages connected to a sugarmoiety without a 2′-modification are phosphorothioate triester linkages.In some embodiments, each internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a modified linkage. In someembodiments, each internucleotidic linkage connected to a sugar moietywithout a 2′-modification is a linkage having the structure of formulaI. In some embodiments, each internucleotidic linkage connected to asugar moiety without a 2′-modification is phosphorothioate linkage. Insome embodiments, each internucleotidic linkage connected to a sugarmoiety without a 2′-modification is a substituted phosphorothioatelinkage. In some embodiments, each internucleotidic linkage connected toa sugar moiety without a 2′-modification is a phosphorothioate triesterlinkage.

In some embodiments, a sugar moiety without a 2′-modification is a sugarmoiety found in a natural DNA nucleoside.

In some embodiments, for a wing-core-wing structure, the 5′-end wingcomprises only one chiral internucleotidic linkage. In some embodiments,for a wing-core-wing structure, the 5′-end wing comprises only onechiral internucleotidic linkage at the 5′-end of the wing. In someembodiments, for a wing-core-wing structure, the 3′-end wing comprisesonly one chiral internucleotidic linkage. In some embodiments, for awing-core-wing structure, the 3′-end wing comprises only one chiralinternucleotidic linkage at the 3′-end of the wing. In some embodiments,for a wing-core-wing structure, each wing comprises only one chiralinternucleotidic linkage. In some embodiments, for a wing-core-wingstructure, each wing comprises only one chiral internucleotidic linkage,wherein the 5′-end wing comprises only one chiral internucleotidiclinkage at its 5′-end; and the 3′-end wing comprises only one chiralinternucleotidic linkage at its 3′-end. In some embodiments, the onlychiral internucleotidic linkage in the 5′-wing is Rp. In someembodiments, the only chiral internucleotidic linkage in the 5′-wing isSp. In some embodiments, the only chiral internucleotidic linkage in the3′-wing is Rp. In some embodiments, the only chiral internucleotidiclinkage in the 3′-wing is Sp. In some embodiments, the only chiralinternucleotidic linkage in both the 5′- and the 3′-wings are Sp. Insome embodiments, the only chiral internucleotidic linkage in both the5′- and the 3′-wings are Rp. In some embodiments, the only chiralinternucleotidic linkage in the 5′-wing is Sp, and the only chiralinternucleotidic linkage in the 3′-wing is Rp. In some embodiments, theonly chiral internucleotidic linkage in the 5′-wing is Rp, and the onlychiral internucleotidic linkage in the 3′-wing is Sp.

In some embodiments, a wing comprises two chiral internucleotidiclinkages. In some embodiments, a wing comprises only two chiralinternucleotidic linkages, and one or more natural phosphate linkages.In some embodiments, a wing comprises only two chiral internucleotidiclinkages, and two or more natural phosphate linkages. In someembodiments, a wing comprises only two chiral internucleotidic linkages,and two or more consecutive natural phosphate linkages. In someembodiments, a wing comprises only two chiral internucleotidic linkages,and two consecutive natural phosphate linkages. In some embodiments, awing comprises only two chiral internucleotidic linkages, and threeconsecutive natural phosphate linkages. In some embodiments, a 5′-wing(to a core) comprises only two chiral internucleotidic linkages, one atits 5′-end and the other at its 3′-end, with one or more naturalphosphate linkages in between. In some embodiments, a 5′-wing (to acore) comprises only two chiral internucleotidic linkages, one at its5′-end and the other at its 3′-end, with two or more natural phosphatelinkages in between. In some embodiments, a 3′-wing (to a core)comprises only two chiral internucleotidic linkages, one at its 3′-endand the other at its 3′-end, with one or more natural phosphate linkagesin between. In some embodiments, a 3′-wing (to a core) comprises onlytwo chiral internucleotidic linkages, one at its 3′-end and the other atits 3′-end, with two or more natural phosphate linkages in between.

In some embodiments, a 5′-wing comprises only two chiralinternucleotidic linkages, one at its 5′-end and the other at its3′-end, with one or more natural phosphate linkages in between, and the3′-wing comprise only one internucleotidic linkage at its 3′-end. Insome embodiments, a 5′-wing (to a core) comprises only two chiralinternucleotidic linkages, one at its 5′-end and the other at its3′-end, with two or more natural phosphate linkages in between, and the3′-wing comprise only one internucleotidic linkage at its 3′-end. Insome embodiments, each chiral internucleotidic linkage independently hasits own stereochemistry. In some embodiments, both chiralinternucleotidic linkages in the 5′-wing have the same stereochemistry.In some embodiments, both chiral internucleotidic linkages in the5′-wing have different stereochemistry. In some embodiments, both chiralinternucleotidic linkages in the 5′-wing are Rp. In some embodiments,both chiral internucleotidic linkages in the 5′-wing are Sp. In someembodiments, chiral internucleotidic linkages in the 5′- and 3′-wingshave the same stereochemistry. In some embodiments, chiralinternucleotidic linkages in the 5′- and 3′-wings are Rp. In someembodiments, chiral internucleotidic linkages in the 5′- and 3′-wingsare Sp. In some embodiments, chiral internucleotidic linkages in the 5′-and 3′-wings have different stereochemistry.

In some embodiments, a chiral, modified phosphate linkage is a chiralphosphorothioate linkage, i.e., phosphorothioate internucleotidiclinkage. In some embodiments, a wing region comprises at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% chiral phosphorothioate internucleotidiclinkages. In some embodiments, all chiral, modified phosphate linkagesare chiral phosphorothioate internucleotidic linkages. In someembodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90% chiral phosphorothioate internucleotidic linkages of a wing regionare of the Sp conformation. In some embodiments, at least about 10%chiral phosphorothioate internucleotidic linkages of a wing region areof the Sp conformation. In some embodiments, at least about 20% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 30% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 40% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 50% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 60% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 70% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 80% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 90% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation. In some embodiments, at least about 95% chiralphosphorothioate internucleotidic linkages of a wing region are of theSp conformation.

In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% chiral phosphorothioate internucleotidic linkages of a wingregion are of the Rp conformation. In some embodiments, at least about10% chiral phosphorothioate internucleotidic linkages of a wing regionare of the Rp conformation. In some embodiments, at least about 20%chiral phosphorothioate internucleotidic linkages of a wing region areof the Rp conformation. In some embodiments, at least about 30% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, at least about 40% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, at least about 50% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, at least about 60% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, at least about 70% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, at least about 80% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, at least about 90% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, at least about 95% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation.

In some embodiments, less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% chiral phosphorothioate internucleotidic linkages of a wingregion are of the Rp conformation. In some embodiments, less than about10% chiral phosphorothioate internucleotidic linkages of a wing regionare of the Rp conformation. In some embodiments, less than about 20%chiral phosphorothioate internucleotidic linkages of a wing region areof the Rp conformation. In some embodiments, less than about 30% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, less than about 40% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, less than about 50% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, less than about 60% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, less than about 70% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, less than about 80% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, less than about 90% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, less than about 95% chiralphosphorothioate internucleotidic linkages of a wing region are of theRp conformation. In some embodiments, a wing region has only one Rpchiral phosphorothioate internucleotidic linkages. In some embodiments,a wing region has only one Rp chiral phosphorothioate internucleotidiclinkages, wherein all internucleotide linkages are chiralphosphorothioate internucleotidic linkages.

In some embodiments, a core region has a length of one or more bases. Insome embodiments, a core region has a length of two or more bases. Insome embodiments, a core region has a length of three or more bases. Insome embodiments, a core region has a length of four or more bases. Insome embodiments, a core region has a length of five or more bases. Insome embodiments, a core region has a length of six or more bases. Insome embodiments, a core region has a length of seven or more bases. Insome embodiments, a core region has a length of eight or more bases. Insome embodiments, a core region has a length of nine or more bases. Insome embodiments, a core region has a length of ten or more bases. Insome embodiments, a core region has a length of 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, or more bases. In certain embodiments, a core regionhas a length of 11 or more bases. In certain embodiments, a core regionhas a length of 12 or more bases. In certain embodiments, a core regionhas a length of 13 or more bases. In certain embodiments, a core regionhas a length of 14 or more bases. In certain embodiments, a core regionhas a length of 15 or more bases. In certain embodiments, a core regionhas a length of 16 or more bases. In certain embodiments, a core regionhas a length of 17 or more bases. In certain embodiments, a core regionhas a length of 18 or more bases. In certain embodiments, a core regionhas a length of 19 or more bases. In certain embodiments, a core regionhas a length of 20 or more bases. In certain embodiments, a core regionhas a length of more than 20 bases. In certain embodiments, a coreregion has a length of 2 bases. In certain embodiments, a core regionhas a length of 3 bases. In certain embodiments, a core region has alength of 4 bases. In certain embodiments, a core region has a length of5 bases. In certain embodiments, a core region has a length of 6 bases.In certain embodiments, a core region has a length of 7 bases. Incertain embodiments, a core region has a length of 8 bases. In certainembodiments, a core region has a length of 9 bases. In certainembodiments, a core region has a length of 10 bases. In certainembodiments, a core region has a length of 11 bases. In certainembodiments, a core region has a length of 12 bases. In certainembodiments, a core region has a length of 13 bases. In certainembodiments, a core region has a length of 14 bases. In certainembodiments, a core region has a length of 15 bases. In certainembodiments, a core region has a length of 16 bases. In certainembodiments, a core region has a length of 17 bases. In certainembodiments, a core region has a length of 18 bases. In certainembodiments, a core region has a length of 19 bases. In certainembodiments, a core region has a length of 20 bases.

In some embodiments, a core comprises one or more modifiedinternucleotidic linkages. In some embodiments, a core comprises one ormore natural phosphate linkages. In some embodiments, a coreindependently comprises one or more modified internucleotidic linkagesand one or more natural phosphate linkages. In some embodiments, a corecomprises no natural phosphate linkages. In some embodiments, each corelinkage is a modified internucleotidic linkage.

In some embodiments, a core comprises at least one natural phosphatelinkage. In some embodiments, a core comprises at least two modifiedinternucleotidic linkages. In some embodiments, a core comprises atleast three modified internucleotidic linkages. In some embodiments, acore comprises at least four modified internucleotidic linkages. In someembodiments, a core comprises at least five modified internucleotidiclinkages. In some embodiments, a core comprises at least six modifiedinternucleotidic linkages. In some embodiments, a core comprises atleast seven modified internucleotidic linkages. In some embodiments, acore comprises at least eight modified internucleotidic linkages. Insome embodiments, a core comprises at least nine modifiedinternucleotidic linkages. In some embodiments, a core comprises atleast ten modified internucleotidic linkages. In some embodiments, acore comprises at least 11 modified internucleotidic linkages. In someembodiments, a core comprises at least 12 modified internucleotidiclinkages. In some embodiments, a core comprises at least 13 modifiedinternucleotidic linkages. In some embodiments, a core comprises atleast 14 modified internucleotidic linkages. In some embodiments, a corecomprises at least 15 modified internucleotidic linkages. In someembodiments, a core comprises at least 16 modified internucleotidiclinkages. In some embodiments, a core comprises at least 17 modifiedinternucleotidic linkages. In some embodiments, a core comprises atleast 18 modified internucleotidic linkages. In some embodiments, a corecomprises at least 19 modified internucleotidic linkages. In someembodiments, a core comprises at least 20 modified internucleotidiclinkages.

In some embodiments, a core comprises one or more chiralinternucleotidic linkages. In some embodiments, a core comprises one ormore natural phosphate linkages. In some embodiments, a coreindependently comprises one or more chiral internucleotidic linkages andone or more natural phosphate linkages. In some embodiments, a corecomprises no natural phosphate linkages. In some embodiments, each corelinkage is a chiral internucleotidic linkage.

In some embodiments, a core comprises at least one natural phosphatelinkage. In some embodiments, a core comprises at least two chiralinternucleotidic linkages. In some embodiments, a core comprises atleast three chiral internucleotidic linkages. In some embodiments, acore comprises at least four chiral internucleotidic linkages. In someembodiments, a core comprises at least five chiral internucleotidiclinkages. In some embodiments, a core comprises at least six chiralinternucleotidic linkages. In some embodiments, a core comprises atleast seven chiral internucleotidic linkages. In some embodiments, acore comprises at least eight chiral internucleotidic linkages. In someembodiments, a core comprises at least nine chiral internucleotidiclinkages. In some embodiments, a core comprises at least ten chiralinternucleotidic linkages. In some embodiments, a core comprises atleast 11 chiral internucleotidic linkages. In some embodiments, a corecomprises at least 12 chiral internucleotidic linkages. In someembodiments, a core comprises at least 13 chiral internucleotidiclinkages. In some embodiments, a core comprises at least 14 chiralinternucleotidic linkages. In some embodiments, a core comprises atleast 15 chiral internucleotidic linkages. In some embodiments, a corecomprises at least 16 chiral internucleotidic linkages. In someembodiments, a core comprises at least 17 chiral internucleotidiclinkages. In some embodiments, a core comprises at least 18 chiralinternucleotidic linkages. In some embodiments, a core comprises atleast 19 chiral internucleotidic linkages. In some embodiments, a corecomprises at least 20 chiral internucleotidic linkages.

In some embodiments, a core comprises one natural phosphate linkage. Insome embodiments, a core comprises two chiral internucleotidic linkages.In some embodiments, a core comprises three chiral internucleotidiclinkages. In some embodiments, a core comprises four chiralinternucleotidic linkages. In some embodiments, a core comprises fivechiral internucleotidic linkages. In some embodiments, a core comprisessix chiral internucleotidic linkages. In some embodiments, a corecomprises seven chiral internucleotidic linkages. In some embodiments, acore comprises eight chiral internucleotidic linkages. In someembodiments, a core comprises nine chiral internucleotidic linkages. Insome embodiments, a core comprises ten chiral internucleotidic linkages.In some embodiments, a core comprises 11 chiral internucleotidiclinkages. In some embodiments, a core comprises 12 chiralinternucleotidic linkages. In some embodiments, a core comprises 13chiral internucleotidic linkages. In some embodiments, a core comprises14 chiral internucleotidic linkages. In some embodiments, a corecomprises 15 chiral internucleotidic linkages. In some embodiments, acore comprises 16 chiral internucleotidic linkages. In some embodiments,a core comprises 17 chiral internucleotidic linkages. In someembodiments, a core comprises 18 chiral internucleotidic linkages. Insome embodiments, a core comprises 19 chiral internucleotidic linkages.In some embodiments, a core comprises 20 chiral internucleotidiclinkages.

In some embodiments, a core region has a pattern of backbone chiralcenters comprising (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or(Sp)t(Rp)n(Sp)m, wherein each of m, n, t and Np is independently asdefined and described in the present disclosure. In some embodiments, acore region has a pattern of backbone chiral centers comprising(Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In someembodiments, a core region has a pattern of backbone chiral centerscomprising (Sp)m(Rp)n. In some embodiments, a core region has a patternof backbone chiral centers comprising (Sp)m(Rp)n, wherein m>2 and nis 1. In some embodiments, a core region has a pattern of backbonechiral centers comprising (Rp)n(Sp)m. In some embodiments, a core regionhas a pattern of backbone chiral centers comprising (Rp)n(Sp)m, whereinm>2 and n is 1. In some embodiments, a core region has a pattern ofbackbone chiral centers comprising (Np)t(Rp)n(Sp)m. In some embodiments,a core region has a pattern of backbone chiral centers comprising(Np)t(Rp)n(Sp)m, wherein m>2 and n is 1. In some embodiments, a coreregion has a pattern of backbone chiral centers comprising(Np)t(Rp)n(Sp)m, wherein t>2, m>2 and n is 1. In some embodiments, acore region has a pattern of backbone chiral centers comprising(Sp)t(Rp)n(Sp)m. In some embodiments, a core region has a pattern ofbackbone chiral centers comprising (Sp)t(Rp)n(Sp)m, wherein m>2 and nis 1. In some embodiments, a core region has a pattern of backbonechiral centers comprising (Sp)t(Rp)n(Sp)m, wherein t>2, m>2 and n is 1.Among other things, the present disclosure demonstrates that, in someembodiments, such patterns can provide and/or enhance controlledcleavage, improved cleavage rate, selectivity, etc., of a targetsequence, e.g., an RNA sequence. Example patterns of backbone chiralcenters are described in the present disclosure.

In some embodiments, at least 60% of the chiral internucleotidiclinkages in the core region are Sp. In some embodiments, at least 65% ofthe chiral internucleotidic linkages in the core region are Sp. In someembodiments, at least 66% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 67% of the chiralinternucleotidic linkages in the core region are Sp. In someembodiments, at least 70% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 75% of the chiralinternucleotidic linkages in the core region are Sp. In someembodiments, at least 80% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 85% of the chiralinternucleotidic linkages in the core region are Sp. In someembodiments, at least 90% of the chiral internucleotidic linkages in thecore region are Sp. In some embodiments, at least 95% of the chiralinternucleotidic linkages in the core region are Sp.

In some embodiments, a wing-core-wing (i.e., X—Y—X) motif is representednumerically as, e.g., 5-10-4, meaning the wing to the 5′-end of the coreis 5 bases in length, the core region is 10 bases in length, and thewing region to the 3′-end of the core is 4-bases in length. In someembodiments, a wing-core-wing motif is any of, e.g. 2-16-2, 3-14-3,4-12-4, 5-10-5, 2-9-6, 3-9-3, 3-9-4, 3-9-5, 4-7-4, 4-9-3, 4-9-4, 4-9-5,4-10-5, 4-11-4, 4-11-5, 5- 7-5, 5-8-6, 8-7-5, 7-7-6, 5-9-3, 5-9-5,5-10-4, 5-10-5, 6-7-6, 6-8-5, and 6-9-2, etc. In certain embodiments, awing-core-wing motif is 5-10-5. In certain embodiments, a wing-core-wingmotif is 7-7-6. In certain embodiments, a wing-core-wing motif is 8-7-5.

In some embodiments, a wing-core motif is 5-15, 6-14, 7-13, 8-12, 9-12,etc. In some embodiments, a core-wing motif is 5-15, 6-14, 7-13, 8-12,9-12, etc.

In some embodiments, the internucleosidic linkages of providedoligonucleotides of such wing-core-wing (i.e., X—Y—X) motifs are allchiral, modified phosphate linkages. In some embodiments, theinternucleosidic linkages of provided oligonucleotides of suchwing-core-wing (i.e., X—Y—X) motifs are all chiral phosphorothioateinternucleotidic linkages. In some embodiments, chiral internucleotidiclinkages of provided oligonucleotides of such wing-core-wing motifs areat least about 10, 20, 30, 40, 50, 50, 70, 80, or 90% chiral, modifiedphosphate internucleotidic linkages. In some embodiments, chiralinternucleotidic linkages of provided oligonucleotides of suchwing-core-wing motifs are at least about 10, 20, 30, 40, 50, 60, 70, 80,or 90% chiral phosphorothioate internucleotidic linkages. In someembodiments, chiral internucleotidic linkages of providedoligonucleotides of such wing-core-wing motifs are at least about 10,20, 30, 40, 50, 50, 70, 80, or 90% chiral phosphorothioateinternucleotidic linkages of the Sp conformation.

In some embodiments, each wing region of a wing-core-wing motifoptionally contains chiral, modified phosphate internucleotidiclinkages. In some embodiments, each wing region of a wing-core-wingmotif optionally contains chiral phosphorothioate internucleotidiclinkages. In some embodiments, each wing region of a wing-core-wingmotif contains chiral phosphorothioate internucleotidic linkages. Insome embodiments, the two wing regions of a wing-core-wing motif havethe same internucleotidic linkage stereochemistry. In some embodiments,the two wing regions have different internucleotidic linkagestereochemistry. In some embodiments, each internucleotidic linkage inthe wings is independently a chiral internucleotidic linkage.

In some embodiments, the core region of a wing-core-wing motifoptionally contains chiral, modified phosphate internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifoptionally contains chiral phosphorothioate internucleotidic linkages.In some embodiments, the core region of a wing-core-wing motif comprisesa repeating pattern of internucleotidic linkage stereochemistry. In someembodiments, the core region of a wing-core-wing motif has a repeatingpattern of internucleotidic linkage stereochemistry. In someembodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)mRp or Rp(Sp)m, wherein in is 1-50. In someembodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)mRp or Rp(Sp)m, wherein m is 1-50. In someembodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is (Sp)mRp, wherein m is 1-50. In someembodiments, the core region of a wing-core-wing motif comprisesrepeating pattern of internucleotidic linkage stereochemistry, whereinthe repeating pattern is Rp(Sp)m, wherein m is 1-50. In someembodiments, the core region of a wing-core-wing motif has repeatingpattern of internucleotidic linkage stereochemistry, wherein therepeating pattern is (Sp)mRp or Rp(Sp)m, wherein m is 1-50. In someembodiments, the core region of a wing-core-wing motif has repeatingpattern of internucleotidic linkage stereochemistry, wherein therepeating pattern is (Sp)mRp, wherein m is 1-50. In some embodiments,the core region of a wing-core-wing motif has repeating pattern ofinternucleotidic linkage stereochemistry, wherein the repeating patternis Rp(Sp)m, wherein m is 1-50. In some embodiments, the core region of awing-core-wing motif has repeating pattern of internucleotidic linkagestereochemistry, wherein the repeating pattern is a motif comprising atleast 33% of internucleotidic linkage in the S conformation. In someembodiments, the core region of a wing-core-wing motif has repeatingpattern of internucleotidic linkage stereochemistry, wherein therepeating pattern is a motif comprising at least 50% of internucleotidiclinkage in the S conformation. In some embodiments, the core region of awing-core-wing motif has repeating pattern of internucleotidic linkagestereochemistry, wherein the repeating pattern is a motif comprising atleast 66% of internucleotidic linkage in the S conformation. In someembodiments, the core region of a wing-core-wing motif has repeatingpattern of internucleotidic linkage stereochemistry, wherein therepeating pattern is a repeating triplet motif selected from RpRpSp andSpSpRp. In some embodiments, the core region of a wing-core-wing motifhas repeating pattern of internucleotidic linkage stereochemistry,wherein the repeating pattern is a repeating RpRpSp. In someembodiments, the core region of a wing-core-wing motif has repeatingpattern of internucleotidic linkage stereochemistry, wherein therepeating pattern is a repeating SpSpRp.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers in the core region comprises (Sp)mRpor Rp(Sp)m. In some embodiments, the present disclosure provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers in the core regioncomprises Rp(Sp)m. In some embodiments, the present disclosure providesa chirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers in the core regioncomprises (Sp)mRp. In some embodiments, m is 2. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises Rp(Sp)₂. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Sp)₂Rip(Sp)₂. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Rp)₂Rp(Sp)₂. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises RpSpRp(Sp)₂. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises SpRpRp(Sp)₂. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Sp)Rp.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Sp)mRp or Rp(Sp)m. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises Rp(Sp)m. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters comprises (Sp)mRp. In some embodiments, m is 2. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises Rp(Sp)₂. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters comprises (Sp)₂Rp(Sp)₂. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centerscomprises (Rp)₂Rp(Sp)₂. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers comprisesRpSpRp(Sp)₂. In some embodiments, the present disclosure provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers comprises SpRpRp(Sp)₂. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Sp)₂Rp.

As defined herein, m is 1-50. In some embodiments, m is 1. In someembodiments, m is 2-50. In some embodiments, in is 2, 3, 4, 5, 6, 7 or8, In some embodiments, m is 3, 4, 5, 6, 7 or 8. In some embodiments, mis 4, 5, 6, 7 or 8. In some embodiments, m is 5, 6, 7 or 8. In someembodiments, m is 6, 7 or 8. In some embodiments, m is 7 or 8, In someembodiments, m is 2. In some embodiments, m is 3. In some embodiments, mis 4. In some embodiments, m is 5. In some embodiments, m is 6. In someembodiments, m is 7. In some embodiments, m is 8. In some embodiments, mis 9. In some embodiments, m is 10. In some embodiments, m is 11. Insome embodiments, m is 12. In some embodiments, m is 13. In someembodiments, m is 14. In some embodiments, m is 15. In some embodiments,m is 16. In some embodiments, m is 17. In some embodiments, m is 18. Insome embodiments, m is 19. In some embodiments, m is 20. In someembodiments, m is 21, In some embodiments, m is 22. In some embodiments,m is 23. In some embodiments, m is 24. In some embodiments, m is 25. Insome embodiments, m is greater than 25.

In some embodiments, a repeating pattern is (Sp)m(Rp)n, wherein n is1-10, and m is independently as defined above and described herein. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Sp)m(Rp)n. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Sp)m(Rp)n. In some embodiments, arepeating pattern is (Rp)n(Sp)m, wherein n is 1-10, and m isindependently as defined above and described herein. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Rp)n(Sp)m. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Rp)n(Sp)m. In some embodiments,(Rp)n(Sp)m is (Rp)(Sp)₂. In some embodiments, (Sp)n(Rp)m is (Sp)₂(Rp).

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide composition of an oligonucleotide type whosepattern of backbone chiral centers comprises (Sp)m(Rp)n(Sp)t. In someembodiments, a repeating pattern is (Sp)m(Rp)n(Sp)t, wherein n is 1-10,t is 1-50, and m is as defined above and described herein. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers in the core region comprises (Sp)m(Rp)n(Sp)t. Insome embodiments, a repeating pattern is (Sp)t(Rp)n(Sp)m, wherein n is1-10, t is 1-50, and m is as defined above and described herein. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide composition of an oligonucleotide type whose pattern ofbackbone chiral centers comprises (Sp)t(Rp)n(Sp)m. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters in the core region comprises (Sp)t(Rp)n(Sp)m.

In some embodiments, a repeating pattern is (Np)t(Rp)n(Sp)m, wherein nis 1-10, t is 1-50, Np is independently Rp or Sp, and m is as definedabove and described herein. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide composition of anoligonucleotide type whose pattern of backbone chiral centers comprises(Np)t(Rp)n(Sp)m. In some embodiments, the present disclosure provides achirally controlled oligonucleotide composition of an oligonucleotidetype whose pattern of backbone chiral centers in the core regioncomprises (Np)t(Rp)n(Sp)m. In some embodiments, a repeating pattern is(Np)m(Rp)n(Sp)t, wherein n is 1-10, t is 1-50, Np is independently Rp orSp, and m is as defined above and described herein. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomposition of an oligonucleotide type whose pattern of backbone chiralcenters comprises (Np)m(Rp)n(Sp)t. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide composition ofan oligonucleotide type whose pattern of backbone chiral centers in thecore region comprises (Np)m(Rp)n(Sp)t. In some embodiments, Np is Rp. Insome embodiments, Np is Sp. In some embodiments, all Np are the same. Insome embodiments, all Np are Sp. In some embodiments, at least one Np isdifferent from the other Np. In some embodiments, t is 2.

As defined herein, n is 1-10. In some embodiments, n is 1, 2, 3, 4, 5,6, 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2, 3,4, 5, 6, 7 or 8. In some embodiments, n is 3, 4, 5, 6, 7 or 8. In someembodiments, n is 4, 5, 6, 7 or 8. In some embodiments, n is 5, 6, 7 or8. In some embodiments, n is 6, 7 or 8. In some embodiments, n is 7 or8. In some embodiments, n is 1. In some embodiments, n is 2. In someembodiments, n is 3. In some embodiments, n is 4. In some embodiments, nis 5. In some embodiments, n is 6. In some embodiments, n is 7. In someembodiments, n is 8. In some embodiments, n is 9. In some embodiments, nis 10.

As defined herein, t is 1-50. In some embodiments, t is 1. In someembodiments, t is 2-50. In some embodiments, t is 2, 3, 4, 5, 6, 7 or 8.In some embodiments, t is 3, 4, 5, 6, 7 or 8. In some embodiments, t is4, 5, 6, 7 or 8. In some embodiments, t is 5, 6, 7 or 8. In someembodiments, t is 6, 7 or 8. In some embodiments, t is 7 or 8. In someembodiments, t is 2. In some embodiments, t is 3. In some embodiments, tis 4. In some embodiments, t is 5. In some embodiments, t is 6. In someembodiments, t is 7. In some embodiments, t is 8. In some embodiments, tis 9. In some embodiments, t is 10. In some embodiments, t is 11. Insome embodiments, t is 12. In some embodiments, t is 13. In someembodiments, t is 14. In some embodiments, t is 15. In some embodiments,t is 16. In some embodiments, t is 17. In some embodiments, t is 18. Insome embodiments, t is 19. In some embodiments, t is 20. In someembodiments, t is 21, In some embodiments, t is 22, In some embodiments,t is 23, In some embodiments, t is 24. In some embodiments, t is 25. Insome embodiments, t is greater than 25.

In some embodiments, at least one of m and t is greater than 2. In someembodiments, at least one of m and t is greater than 3. In someembodiments, at least one of in and t is greater than 4. In someembodiments, at least one of m and t is greater than 5. In someembodiments, at least one of m and t is greater than 6. In someembodiments, at least one of m and t is greater than 7. In someembodiments, at least one of m and t is greater than 8. In someembodiments, at least one of m and t is greater than 9. In someembodiments, at least one of m and t is greater than 10. In someembodiments, at least one of m and t is greater than 11. In someembodiments, at least one of in and t is greater than 12. In someembodiments, at least one of m and t is greater than 13. In someembodiments, at least one of m and t is greater than 14. In someembodiments, at least one of m and t is greater than 15. In someembodiments, at least one of m and t is greater than 16. In someembodiments, at least one of m and t is greater than 17. In someembodiments, at least one of m and t is greater than 18. In someembodiments, at least one of m and t is greater than 19. In someembodiments, at least one of m and t is greater than 20. In someembodiments, at least one of m and t is greater than 21, In someembodiments, at least one of m and t is greater than 22. In someembodiments, at least one of m and t is greater than 23. In someembodiments, at least one of m and t is greater than 24. In someembodiments, at least one of m and t is greater than 25.

In some embodiments, each one of m and t is greater than 2. In someembodiments, each one of m and t is greater than 3. In some embodiments,each one of m and t is greater than 4. In some embodiments, each one ofm and t is greater than 5. In some embodiments, each one of m and t isgreater than 6, In some embodiments, each one of nm and t is greaterthan 7. In some embodiments, each one of m and t is greater than 8. Insome embodiments, each one of m and t is greater than 9. In someembodiments, each one of m and t is greater than 10. In someembodiments, each one of m and t is greater than 11. In someembodiments, each one of m and t is greater than 12. In someembodiments, each one of m and t is greater than 13. In someembodiments, each one of m and t is greater than 14. In someembodiments, each one of m and t is greater than 15. In someembodiments, each one of m and t is greater than 16, In someembodiments, each one of m and t is greater than 17. In someembodiments, each one of m and t is greater than 18. In someembodiments, each one of m and t is greater than 19. In someembodiments, each one of nm and t is greater than 20.

In some embodiments, the sum of m and t is greater than 3. In someembodiments, the sum of m and t is greater than 4. In some embodiments,the sum of m and t is greater than 5. In some embodiments, the sum of inand t is greater than 6. In some embodiments, the sum of m and t isgreater than 7. In some embodiments, the sum of m and t is greater than8. In some embodiments, the sum of m and t is greater than 9. In someembodiments, the sum of m and t is greater than 10. In some embodiments,the sum of m and t is greater than 11. In some embodiments, the sum of mand t is greater than 12. In some embodiments, the sum of m and t isgreater than 13. In some embodiments, the sum of m and t is greater than14. In some embodiments, the sum of m and t is greater than 15. In someembodiments, the sum of m and t is greater than 16. In some embodiments,the sum of m and t is greater than 17. In some embodiments, the sum of mand t is greater than 18. In some embodiments, the sum of m and t isgreater than 19. In some embodiments, the sum of m and t is greater than20. In some embodiments, the sum of m and t is greater than 21. In someembodiments, the sum of m and t is greater than 22. In some embodiments,the sum of m and t is greater than 23. In some embodiments, the sum of iand t is greater than 24. In some embodiments, the sum of m and t isgreater than 25.

In some embodiments, n is 1, and at least one of m and t is greaterthan 1. In some embodiments, n is 1 and each of m and t is independentlygreater than 1. In some embodiments, m>n and t>n. In some embodiments,(Sp)m(Rp)n(Sp)t is (Sp)₂Rp(Sp)₂. In some embodiments, (Sp)t(Rp)n(Sp)m is(Sp)₂Rp(Sp)₂. In some embodiments, (Sp)t(Rp)n(Sp)m is SpRp(Sp)₂. In someembodiments, (Np)t(Rp)n(Sp)m is (Np)tRp(Sp)m. In some embodiments,(Np)t(Rp)n(Sp)m is (Np)₂Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is(Rp)₂Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is (Sp)₂Rp(Sp)m. Insome embodiments, (Np)t(Rp)n(Sp)m is RpSpRp(Sp)m. In some embodiments,(Np)t(Rp)n(Sp)m is SpRpRp(Sp)m.

In some embodiments, (Sp)t(Rp)n(Sp)m is SpRpSpSp. In some embodiments,(Sp)t(Rp)n(Sp)m is (Sp)₂Rp(Sp)₂. In some embodiments, (Sp)t(Rp)n(Sp)m is(Sp)₃Rp(Sp)₃. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)₄Rp(Sp)₄. Insome embodiments, (Sp)t(Rp)n(Sp)m is (Sp)tRp(Sp)₅. In some embodiments,(Sp)t(Rp)n(Sp)m is SpRp(Sp)₅. In some embodiments, (Sp)t(Rp)n(Sp)m is(Sp)₂Rp(Sp)₅. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)₃Rp(Sp)₅. Insome embodiments, (Sp)t(Rp)n(Sp)m is (Sp)₄Rp(Sp)₅. In some embodiments,(Sp)t(Rp)n(Sp)m is (Sp)₅Rp(Sp)₅.

In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)₂Rp(Sp)₂. In someembodiments, (Sp)m(Rp)n(Sp)t is (Sp)₃Rp(Sp)₃. In some embodiments,(Sp)m(Rp)n(Sp)t is (Sp)₄Rp(Sp)₄. In some embodiments, (Sp)m(Rp)n(Sp)t is(Sp)mRp(Sp)₅. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)₂Rp(Sp)₅. Insome embodiments, (Sp)m(Rp)n(Sp)t is (Sp)₃Rp(Sp)₅. In some embodiments,(Sp)m(Rp)n(Sp)t is (Sp)₄Rp(Sp)₅. In some embodiments, (Sp)m(Rp)n(Sp)t is(Sp)₅Rp(Sp)₅.

In some embodiments, the core region comprises at least one Rpinternucleotidic linkage. In some embodiments, the core region of awing-core-wing motif comprises at least one Rp internucleotidic linkage.In some embodiments, a core region comprises at least one Rpphosphorothioate internucleotidic linkage. In some embodiments, the coreregion of a wing-core-wing motif comprises at least one Rpphosphorothioate internucleotidic linkage. In some embodiments, the coreregion of a wing-core-wing motif comprises only one Rp phosphorothioateinternucleotidic linkage. In some embodiments, a core region motifcomprises at least two Rp internucleotidic linkages. In someembodiments, the core region of a wing-core-wing motif comprises atleast two Rp internucleotidic linkages. In some embodiments, the coreregion of a wing-core-wing motif comprises at least two Rpphosphorothioate internucleotidic linkages. In some embodiments, a coreregion comprises at least three Rp internucleotidic linkages. In someembodiments, the core region of a wing-core-wing motif comprises atleast three Rp internucleotidic linkages. In some embodiments, the coreregion comprises at least three Rp phosphorothioate internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifcomprises at least three Rp phosphorothioate internucleotidic linkages.In some embodiments, a core region comprises at least 4, 5, 6, 7, 8, 9,or 10 Rp internucleotidic linkages. In some embodiments, the core regionof a wing-core-wing motif comprises at least 4, 5, 6, 7, 8, 9, or 10 Rpinternucleotidic linkages. In some embodiments, a core region comprisesat least 4, 5, 6, 7, 8, 9, or 10 Rp phosphorothioate internucleotidiclinkages. In some embodiments, the core region of a wing-core-wing motifcomprises at least 4, 5, 6, 7, 8, 9, or 10 Rp phosphorothioateinternucleotidic linkages.

In certain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues at each wing region are 2′-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-OR¹-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-MOE-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-OMe-modified residues. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues in the core region are 2′-deoxyribonucleoside residues. Incertain embodiments, a wing-core-wing motif is a 5-10-5 motif, whereinall internucleotidic linkages are phosphorothioate linkages. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif, wherein allinternucleotidic linkages are chiral phosphorothioate linkages. Incertain embodiments, a wing-core-wing motif is a 5-10-5 motif whereinthe residues at each wing region are 2′-modified residues, the residuesin the core region are 2′-deoxyribonucleoside residues, and allinternucleotidic linkages in the core region are chiral phosphorothioatelinkages. In certain embodiments, a wing-core-wing motif is a 5-10-5motif wherein the residues at each wing region are 2′-OR-modifiedresidues, the residues in the core region are 2′-deoxyribonucleosideresidues, and all internucleotidic linkages in the core region arechiral phosphorothioate linkages. In certain embodiments, awing-core-wing motif is a 5-10-5 motif wherein the residues at each wingregion are 2′-MOE-modified residues, the residues in the core region are2′-deoxyribonucleoside residues, and all internucleotidic linkages inthe core region are chiral phosphorothioate linkages. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif wherein theresidues at each wing region are 2′-OMe-modified residues, the residuesin the core region are 2′-deoxyribonucleoside residues, and allinternucleotidic linkages in the core region are chiral phosphorothioatelinkages.

In some embodiments, residues at the “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core motif is amotif wherein the residues at the “X” wing region are not2′-MOE-modified residues. In certain embodiments, a core-wing motif is amotif wherein the residues at the “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core-wing motifis a motif wherein the residues at each “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core-wing motifis a 5-10-5 motif wherein the residues at each “X” wing region are not2′-MOE-modified residues. In certain embodiments, a wing-core-wing motifis a 5-10-5 motif wherein the residues in the core “Y” region are2′-deoxyribonucleoside residues. In certain embodiments, awing-core-wing motif is a 5-10-5 motif, wherein all internucleotidiclinkages are phosphorothioate internucleotidic linkages. In certainembodiments, a wing-core-wing motif is a 5-10-5 motif, wherein allinternucleotidic linkages are chiral phosphorothioate internucleotidiclinkages. In certain embodiments, a wing-core-wing motif is a 5-10-5motif wherein the residues at each “X” wing region are not2′-MOE-modified residues, the residues in the core “Y” region are2′-deoxyribonucleoside, and all internucleotidic linkages are chiralphosphorothioate internucleotidic linkages.

In some embodiments, a chiral, modified phosphate linkage is a chiralphosphorothioate linkage, i.e., phosphorothioate internucleotidiclinkage. In some embodiments, a core region comprises at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% chiral phosphorothioate internucleotidiclinkages. In some embodiments, all chiral, modified phosphate linkagesare chiral phosphorothioate internucleotidic linkages. In someembodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90% chiral phosphorothioate internucleotidic linkages of a core regionare of the Sp conformation. In some embodiments, at least about 10%chiral phosphorothioate internucleotidic linkages of a core region areof the Sp conformation. In some embodiments, at least about 20% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 30% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 40% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 50% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 60% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 70% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 80% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 90% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation. In some embodiments, at least about 95% chiralphosphorothioate internucleotidic linkages of a core region are of theSp conformation.

In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% chiral phosphorothioate internucleotidic linkages of a coreregion are of the Rp conformation. In some embodiments, at least about10% chiral phosphorothioate internucleotidic linkages of a core regionare of the Rp conformation. In some embodiments, at least about 20%chiral phosphorothioate internucleotidic linkages of a core region areof the Rp conformation. In some embodiments, at least about 30% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, at least about 40% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, at least about 50% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, at least about 60% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, at least about 70% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, at least about 80% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, at least about 90% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, at least about 95% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation.

In some embodiments, less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% chiral phosphorothioate internucleotidic linkages of a coreregion are of the Rp conformation. In some embodiments, less than about10% chiral phosphorothioate internucleotidic linkages of a core regionare of the Rp conformation. In some embodiments, less than about 20%chiral phosphorothioate internucleotidic linkages of a core region areof the Rp conformation. In some embodiments, less than about 30% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, less than about 40% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, less than about 50% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, less than about 60% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, less than about 70% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, less than about 80% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, less than about 90% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, less than about 95% chiralphosphorothioate internucleotidic linkages of a core region are of theRp conformation. In some embodiments, a core region has only one Rpchiral phosphorothioate internucleotidic linkages. In some embodiments,a core region has only one Rp chiral phosphorothioate internucleotidiclinkages, wherein all internucleotide linkages are chiralphosphorothioate internucleotidic linkages.

As understood by a person having ordinary skill in the art, providedoligonucleotides and compositions, among other things, can target agreat number of nucleic acid polymers. For instance, in someembodiments, provided oligonucleotides and compositions may target atranscript of a nucleic acid sequence, wherein a common base sequence ofoligonucleotides (e.g., a base sequence of an oligonucleotide type)comprises or is a sequence complementary to a sequence of thetranscript. In some embodiments, a common base sequence comprises asequence complimentary to a sequence of a target. In some embodiments, acommon base sequence is a sequence complimentary to a sequence of atarget. In some embodiments, a common base sequence comprises or is asequence 100% complimentary to a sequence of a target. In someembodiments, a common base sequence comprises a sequence 100%complimentary to a sequence of a target. In some embodiments, a commonbase sequence is a sequence 100% complimentary to a sequence of atarget. In some embodiments, a common base sequence in a core comprisesor is a sequence complimentary to a sequence of a target. In someembodiments, a common base sequence in a core comprises a sequencecomplimentary to a sequence of a target. In some embodiments, a commonbase sequence in a core is a sequence % complimentary to a sequence of atarget. In some embodiments, a common base sequence in a core comprisesor is a sequence 100% complimentary to a sequence of a target. In someembodiments, a common base sequence in a core comprises a sequence 100%complimentary to a sequence of a target. In some embodiments, a commonbase sequence in a core is a sequence 100% complimentary to a sequenceof a target.

In some embodiments, as described in this disclosure, providedoligonucleotides and compositions may provide new cleavage patterns,higher cleavage rate, higher cleavage degree, higher cleavageselectivity, etc. In some embodiments, provided compositions canselectively suppress (e.g., cleave) a transcript from a target nucleicacid sequence which has one or more similar sequences exist within asubject or a population, each of the target and its similar sequencescontains a specific nucleotidic characteristic sequence element thatdefines the target sequence relative to the similar sequences. In someembodiments, for example, a target sequence is a wild-type allele orcopy of a gene, and a similar sequence is a sequence has very similarbase sequence, e.g., a sequence having SNP, mutations, etc.; In someembodiments, a characteristic sequence element defines that targetsequence relative to the similar sequence: for example, when a targetsequence is a Huntington's disease-causing allele with T at rs362307 (Uin the corresponding RNA; C for the non-disease-causing allele), acharacteristic sequence comprises this SNP.

In some embodiments, a similar sequence has greater than 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence homology with a target sequence. In some embodiments, a targetsequence is a disease-causing copy of a nucleic acid sequence comprisingone or more mutations and/or SNPs, and a similar sequence is a copy notcausing the disease (wild type). In some embodiments, a target sequencecomprises a mutation, wherein a similar sequence is the correspondingwild-type sequence. In some embodiments, a target sequence is a mutantallele, while a similar sequence is a wild-type allele. In someembodiments, a target sequence comprises an SNP that is associated witha disease-causing allele, while a similar sequence comprises the sameSNP that is not associates with the disease-causing allele. In someembodiments, the region of a target sequence that is complementary to acommon base sequence of a provided oligonucleotide composition hasgreater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence homology with the corresponding region ofa similar sequence. In some embodiments, the region of a target sequencethat is complementary to a common base sequence of a providedoligonucleotide composition differs from the corresponding region of asimilar sequence at less than 5, less than 4, less than 3, less than 2,or only 1 base pairs. In some embodiments, the region of a targetsequence that is complementary to a common base sequence of a providedoligonucleotide composition differs from the corresponding region of asimilar sequence only at a mutation site or SNP site. In someembodiments, the region of a target sequence that is complementary to acommon base sequence of a provided oligonucleotide composition differsfrom the corresponding region of a similar sequence only at a mutationsite. In some embodiments, the region of a target sequence that iscomplementary to a common base sequence of a provided oligonucleotidecomposition differs from the corresponding region of a similar sequenceonly at an SNP site.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a characteristic sequence element. In some embodiments,a common base sequence comprises a sequence complementary to acharacteristic sequence element. In some embodiments, a common basesequence is a sequence complementary to a characteristic sequenceelement. In some embodiments, a common base sequence comprises or is asequence 100% complementary to a characteristic sequence element. Insome embodiments, a common base sequence comprises a sequence 100%complementary to a characteristic sequence element. In some embodiments,a common base sequence is a sequence 100% complementary to acharacteristic sequence element. In some embodiments, a common basesequence in a core comprises or is a sequence complementary to acharacteristic sequence element. In some embodiments, a common basesequence in a core comprises a sequence complementary to acharacteristic sequence element. In some embodiments, a common basesequence in a core is a sequence complementary to a characteristicsequence element. In some embodiments, a common base sequence in a corecomprises or is a sequence 100% complementary to a characteristicsequence element. In some embodiments, a common base sequence in a corecomprises a sequence 100% complementary to a characteristic sequenceelement. In some embodiments, a common base sequence in a core is asequence 100% complementary to a characteristic sequence element.

In some embodiments, a characteristic sequence element comprises or is amutation. In some embodiments, a characteristic sequence elementcomprises a mutation. In some embodiments, a characteristic sequenceelement is a mutation. In some embodiments, a characteristic sequenceelement comprises or is a point mutation. In some embodiments, acharacteristic sequence element comprises a point mutation. In someembodiments, a characteristic sequence element is a point mutation. Insome embodiments, a characteristic sequence element comprises or is anSNP. In some embodiments, a characteristic sequence element comprises anSNP. In some embodiments, a characteristic sequence element is an SNP.

In some embodiments, a common base sequence 100% matches a targetsequence, which it does not 100% match a similar sequence of the targetsequence. For example, in some embodiments, a common base sequencematches a mutation in the disease-causing copy or allele of a targetnucleic acid sequence, but does not match a non-disease-causing copy orallele at the mutation site; in some other embodiments, a common basesequence matches an SNP in the disease-causing allele of a targetnucleic acid sequence, but does not match a non-disease-causing alleleat the corresponding site. In some embodiments, a common base sequencein a core 100% matches a target sequence, which it does not 100% match asimilar sequence of the target sequence. For example, in WV-1092, itscommon base sequence (and its common base sequence in its core) matchesthe disease-causing U, but not the non-disease causing (wild-type) C atrs362307.

Among other things, the present disclosure recognizes that a basesequence may have impact on oligonucleotide properties. In someembodiments, a base sequence may have impact on cleavage pattern of atarget when oligonucleotides having the base sequence are utilized forsuppressing a target, e.g., through a pathway involving RNase H: forexample, FIG. 33 demonstrates that structurally similar (allphosphorothioate linkages, all stereorandom) oligonucleotides havedifferent sequences may have different cleavage patterns. In someembodiments, a common base sequence of a non-stereorandomoligonucleotide compositions (e.g., certain oligonucleotide compositionsprovided in the present disclosure) is a base sequence that when appliedto a DNA oligonucleotide composition (e.g., ONT-415) or a stereorandomall-phosphorothioate oligonucleotide composition (e.g., WV-905),cleavage pattern of the DNA (DNA cleavage pattern) and/or thestereorandom all-phosphorothioate (stereorandom cleavage pattern)composition has a cleavage site within or in the vicinity of acharacteristic sequence element. In some embodiments, a cleavage sitewithin or in the vicinity is within a sequence complementary to a coreregion of a common sequence. In some embodiments, a cleavage site withinor in the vicinity is within a sequence 100% complementary to a coreregion of a common sequence.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site within or in the vicinity of a characteristic sequenceelement in its DNA cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site within acharacteristic sequence element in its DNA cleavage pattern. In someembodiments, a common base sequence is a base sequence that has acleavage site in the vicinity of a characteristic sequence element inits DNA cleavage pattern. In some embodiments, a common base sequence isa base sequence that has a cleavage site in the vicinity of a mutationor SNP of a characteristic sequence element in its DNA cleavage pattern.In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of a mutation in its DNA cleavagepattern. In some embodiments, a common base sequence is a base sequencethat has a cleavage site in the vicinity of an SNP in its DNA cleavagepattern.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site within or in the vicinity of a characteristic sequenceelement in its stereorandom cleavage pattern. In some embodiments, acommon base sequence is a base sequence that has a cleavage site withina characteristic sequence element in its stereorandom cleavage pattern.In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of a characteristic sequence element inits stereorandom cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site in the vicinity ofa mutation or SNP of a characteristic sequence element in itsstereorandom cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site in the vicinity ofa mutation in its stereorandom cleavage pattern. In some embodiments, acommon base sequence is a base sequence that has a cleavage site in thevicinity of an SNP in its stereorandom cleavage pattern.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of a mutation of a characteristicsequence element in its DNA and/or stereorandom cleavage pattern. Insome embodiments, a common base sequence is a base sequence that has acleavage site in the vicinity of a mutation in its DNA and/orstereorandom cleavage pattern. In some embodiments, a common basesequence is a base sequence that has a cleavage site in the vicinity ofa mutation in its DNA cleavage pattern. In some embodiments, a cleavagesite in the vicinity of a mutation is at a mutation, i.e., a cleavagesite is at the internucleotidic linkage of a mutated nucleotide (e.g.,if a mutation is at A in the target sequence of GGGACGTCTT (SEQ ID NO:13), the cleavage is between A and C). In some embodiments, a cleavagesite in the vicinity is a cleavage site 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 internucleotidic linkages away from a mutation, where 0 meanscleavage at the mutation site (e.g., if a mutation is at A in the targetsequence of GGGACGTCTT (SEQ ID NO: 13), the cleavage is between A and Cfor 0 internucleotidic linkage away; a cleavage site 1 internucleotidiclinkage away from the mutation is between G and A to the 5′ from themutation or between C and G to the 3′ from the mutation). In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 5′ from a mutation. Insome embodiments, a cleavage site in the vicinity is a cleavage site 0,1, 2, 3, 4, or 5 internucleotidic linkages away to the 3′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0, 1, 2, 3, 4, or 5 internucleotidic linkages away from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0, 1, 2, 3, 4, or 5 internucleotidic linkages away to the5′ from a mutation. In some embodiments, a cleavage site in the vicinityis a cleavage site 0, 1, 2, 3, 4, or 5 internucleotidic linkages away tothe 3′ from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, 2, 3, or 4 internucleotidic linkagesaway from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, 2, 3, or 4 internucleotidic linkagesaway to the 5′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, 2, 3, or 4 internucleotidiclinkages away to the 3′ from a mutation. In some embodiments, a cleavagesite in the vicinity is a cleavage site 0, 1, 2, or 3 internucleotidiclinkages away from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, 2, or 3 internucleotidic linkagesaway to the 5′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, 2, or 3 internucleotidic linkagesaway to the 3′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site 0, 1, or 2 internucleotidic linkagesaway from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, or 2 internucleotidic linkages away tothe 5′ from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0, 1, or 2 internucleotidic linkages away tothe 3′ from a mutation. In some embodiments, a cleavage site in thevicinity is a cleavage site 0 or 1 internucleotidic linkage away from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0 or 1 internucleotidic linkage away to the 5′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0 or 1 internucleotidic linkage away to the 3′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site 0 internucleotidic linkage away from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site oneinternucleotidic linkage away from a mutation. In some embodiments, acleavage site in the vicinity is a cleavage site one internucleotidiclinkage away to the 5′ from a mutation. In some embodiments, a cleavagesite in the vicinity is a cleavage site one internucleotidic linkageaway to the 3′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site two internucleotidic linkages away froma mutation. In some embodiments, a cleavage site in the vicinity is acleavage site two internucleotidic linkages away to the 5′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site two internucleotidic linkages away to the 3′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site three internucleotidic linkages away from a mutation. Insome embodiments, a cleavage site in the vicinity is a cleavage sitethree internucleotidic linkages away to the 5′ from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site threeinternucleotidic linkages away to the 3′ from a mutation. In someembodiments, a cleavage site in the vicinity is a cleavage site fourinternucleotidic linkages away from a mutation. In some embodiments, acleavage site in the vicinity is a cleavage site four internucleotidiclinkages away to the 5′ from a mutation. In some embodiments, a cleavagesite in the vicinity is a cleavage site four internucleotidic linkagesaway to the 3′ from a mutation. In some embodiments, a cleavage site inthe vicinity is a cleavage site five internucleotidic linkages away froma mutation. In some embodiments, a cleavage site in the vicinity is acleavage site five internucleotidic linkages away to the 5′ from amutation. In some embodiments, a cleavage site in the vicinity is acleavage site five internucleotidic linkages away to the 3′ from amutation.

In some embodiments, a common base sequence is a base sequence that hasa cleavage site in the vicinity of an SNP of a characteristic sequenceelement in its DNA and/or stereorandom cleavage pattern. In someembodiments, a common base sequence is a base sequence that has acleavage site in the vicinity of an SNP in its DNA and/or stereorandomcleavage pattern. In some embodiments, a common base sequence is a basesequence that has a cleavage site in the vicinity of an SNP in its DNAcleavage pattern. In some embodiments, a cleavage site in the vicinityof an SNP is at an SNP, i.e., a cleavage site is at the internucleotidiclinkage of a nucleotide at an SNP (e.g., for the target of WV-905,G*G*C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C (SEQ ID NO: 14), which comprisesrUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 15)(rs362307 bolded), the cleavage is between the bolded rU and theunderlined rG immediately after it). In some embodiments, a cleavagesite in the vicinity is a cleavage site 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, Or10 internucleotidic linkages away from an SNP, where 0 means cleavage atan SNP (e.g., for the target of WV-905,G*G*C*A*C*A*A*G*G*G*C*A*C*A*G*A*C*T*T*C (SEQ ID NO: 14), which comprisesrUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 15)(rs362307 bolded), the cleavage is between the bolded rU and theunderlined rG immediately after it for 0 internucleotidic linkage away;a cleavage site 1 internucleotidic linkage away from an SNP is betweenthe rG and rU to the 5′ from the SNP (underlined:rUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 15)), orbetween rG and rC to the 3′-end of the SNP (underlined:rUrUrUrGrGrArArGrUrCrUrGrUrGrCrCrCrUrUrGrUrGrCrCrC (SEQ ID NO: 15))). Insome embodiments, a cleavage site in the vicinity is a cleavage site 0,1, 2, 3, 4, or 5 internucleotidic linkages away from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 5′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 3′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 5′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, 4, or 5 internucleotidic linkages away to the 3′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, or 4 internucleotidic linkages away from an SNP. In some embodiments,a cleavage site in the vicinity is a cleavage site 0, 1, 2, 3, or 4internucleotidic linkages away to the 5′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,3, or 4 internucleotidic linkages away to the 3′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,or 3 internucleotidic linkages away from an SNP. In some embodiments, acleavage site in the vicinity is a cleavage site 0, 1, 2, or 3internucleotidic linkages away to the 5′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, 2,or 3 internucleotidic linkages away to the 3′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, or2 internucleotidic linkages away from an SNP. In some embodiments, acleavage site in the vicinity is a cleavage site 0, 1, or 2internucleotidic linkages away to the 5′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0, 1, or2 internucleotidic linkages away to the 3′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site 0 or 1internucleotidic linkage away from an SNP. In some embodiments, acleavage site in the vicinity is a cleavage site 0 or 1 internucleotidiclinkage away to the 5′ from an SNP. In some embodiments, a cleavage sitein the vicinity is a cleavage site 0 or 1 internucleotidic linkage awayto the 3′ from an SNP. In some embodiments, a cleavage site in thevicinity is a cleavage site 0 internucleotidic linkage away from an SNP.In some embodiments, a cleavage site in the vicinity is a cleavage siteone internucleotidic linkage away from an SNP. In some embodiments, acleavage site in the vicinity is a cleavage site one internucleotidiclinkage away to the 5′ from an SNP. In some embodiments, a cleavage sitein the vicinity is a cleavage site one internucleotidic linkage away tothe 3′ from an SNP. In some embodiments, a cleavage site in the vicinityis a cleavage site two internucleotidic linkages away from an SNP. Insome embodiments, a cleavage site in the vicinity is a cleavage site twointernucleotidic linkages away to the 5′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site twointernucleotidic linkages away to the 3′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site threeinternucleotidic linkages away from an SNP. In some embodiments, acleavage site in the vicinity is a cleavage site three internucleotidiclinkages away to the 5′ from an SNP. In some embodiments, a cleavagesite in the vicinity is a cleavage site three internucleotidic linkagesaway to the 3′ from an SNP. In some embodiments, a cleavage site in thevicinity is a cleavage site four internucleotidic linkages away from anSNP. In some embodiments, a cleavage site in the vicinity is a cleavagesite four internucleotidic linkages away to the 5′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site fourinternucleotidic linkages away to the 3′ from an SNP. In someembodiments, a cleavage site in the vicinity is a cleavage site fiveinternucleotidic linkages away from an SNP. In some embodiments, acleavage site in the vicinity is a cleavage site five internucleotidiclinkages away to the 5′ from an SNP. In some embodiments, a cleavagesite in the vicinity is a cleavage site five internucleotidic linkagesaway to the 3′ from an SNP. For example, FIG. 33 demonstrates thatstereorandom cleavage pattern of the WV-905 sequence has cleavage sitesat the SNP (between CUGU and GCCC), two internucleotidic linkages away(between GUCU and GUGC, and between GUGC and CCUU), threeinternucleotidic linkages away (between UGCC and CUUG); fourinternucleotidic linkages away (between GCCC and UUGU, and AAGU andCUGU), and five internucleotidic linkages away (between CCCU and UGUG).

In some embodiments, a cleavage site within or in the vicinity of acharacteristic sequence element, e.g., in the vicinity of a mutation, anSNP, etc., is a major cleavage site of a DNA and/or stereorandomcleavage pattern. In some embodiments, a cleavage site within or in thevicinity of a characteristic sequence element is a major cleavage siteof a DNA cleavage pattern. In some embodiments, a cleavage site withinor in the vicinity of a characteristic sequence element is a majorcleavage site of a stereorandom cleavage pattern. In some embodiments, acleavage site in the vicinity of a mutation is a major cleavage site ofa DNA cleavage pattern. In some embodiments, a cleavage site in thevicinity of a mutation is a major cleavage site of a stereorandomcleavage pattern. In some embodiments, a cleavage site in the vicinityof an SNP is a major cleavage site of a DNA cleavage pattern. In someembodiments, a cleavage site in the vicinity of an SNP is a majorcleavage site of a stereorandom cleavage pattern. In some embodiments, amajor cleavage site is within a sequence complementary to a core regionof a common sequence. In some embodiments, a major cleavage site iswithin a sequence 100% complementary to a core region of a commonsequence.

In some embodiments, a major cleavage site is a site having the most, orthe second, third, fourth or fifth most cleavage. In some embodiments, amajor cleavage site is a site having the most, or the second, third, orfourth most cleavage. In some embodiments, a major cleavage site is asite having the most, or the second, or third most cleavage. In someembodiments, a major cleavage site is a site having the most or thesecond most cleavage. In some embodiments, a major cleavage site is asite having the most cleavage. In some embodiments, a major cleavagesite is a site having the second most cleavage. In some embodiments, amajor cleavage site is a site having the third most cleavage. In someembodiments, a major cleavage site is a site having the fourth mostcleavage. In some embodiments, a major cleavage site is a site havingthe fifth most cleavage.

In some embodiments, a major cleavage site is a site wherein greaterthan 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of cleavage occurs.In some embodiments, a major cleavage site is a site wherein greaterthan 5% of cleavage occurs. In some embodiments, a major cleavage siteis a site wherein greater than 10% of cleavage occurs. In someembodiments, a major cleavage site is a site wherein greater than 15% ofcleavage occurs. In some embodiments, a major cleavage site is a sitewherein greater than 20% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 25% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 30% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 35% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than40% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 45% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 50% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 55% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 60% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than65% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 70% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 75% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 80% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 85% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than90% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 91% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 92% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 93% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 94% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein greater than95% of cleavage occurs. In some embodiments, a major cleavage site is asite wherein greater than 96% of cleavage occurs. In some embodiments, amajor cleavage site is a site wherein greater than 97% of cleavageoccurs. In some embodiments, a major cleavage site is a site whereingreater than 98% of cleavage occurs. In some embodiments, a majorcleavage site is a site wherein greater than 99% of cleavage occurs. Insome embodiments, a major cleavage site is a site wherein 100% ofcleavage occurs.

In some embodiments, a major cleavage site is a site wherein greaterthan 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of a target iscleaved. In some embodiments, a major cleavage site is a site whereingreater than 5% of a target is cleaved. In some embodiments, a majorcleavage site is a site wherein greater than 10% of a target is cleaved.In some embodiments, a major cleavage site is a site wherein greaterthan 15% of a target is cleaved. In some embodiments, a major cleavagesite is a site wherein greater than 20% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 25% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 30% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 35% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 40% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 45% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 50% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 55% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 60% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 65% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 70% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 75% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 80% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 85% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 90% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 91% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 92% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 93% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 94% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 95% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 96% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 97% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein greater than 98% of a target is cleaved. In someembodiments, a major cleavage site is a site wherein greater than 99% ofa target is cleaved. In some embodiments, a major cleavage site is asite wherein 100% of a target is cleaved. In some embodiments, acleavage pattern may not have a major cleavage site as no site reachesan absolute cleavage threshold level.

As a person having ordinary skill in the art understands, providedoligonucleotide compositions and methods have various uses as known by aperson having ordinary skill in the art. Methods for assessing providedcompositions, and properties and uses thereof, are also widely known andpracticed by a person having ordinary skill in the art. Exampleproperties, uses, and/or methods include but are not limited to thosedescribed in WO/2014/012081 and WO/2015/107425.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a nucleic acid sequence. In some embodiments, a commonbase sequence comprises or is a sequence 100% complementary to a nucleicacid sequence. In some embodiments, a common base sequence comprises oris a sequence complementary to a disease-causing nucleic acid sequence.In some embodiments, a common base sequence comprises or is a sequence100% complementary to a disease-causing nucleic acid sequence. In someembodiments, a common base sequence comprises or is a sequencecomplementary to a characteristic sequence element of disease-causingnucleic acid sequence, which characteristic sequences differentiate adisease-causing nucleic acid sequence from a non-disease-causing nucleicacid sequence. In some embodiments, a common base sequence comprises oris a sequence 100% complementary to a characteristic sequence element ofdisease-causing nucleic acid sequence, which characteristic sequencesdifferentiate a disease-causing nucleic acid sequence from anon-disease-causing nucleic acid sequence. In some embodiments, a commonbase sequence comprises or is a sequence complementary to adisease-associated nucleic acid sequence. In some embodiments, a commonbase sequence comprises or is a sequence 100% complementary to adisease-associated nucleic acid sequence. In some embodiments, a commonbase sequence comprises or is a sequence complementary to acharacteristic sequence element of disease-associated nucleic acidsequence, which characteristic sequences differentiate adisease-associated nucleic acid sequence from a non-disease-associatednucleic acid sequence. In some embodiments, a common base sequencecomprises or is a sequence 100% complementary to a characteristicsequence element of disease-associated nucleic acid sequence, whichcharacteristic sequences differentiate a disease-associated nucleic acidsequence from a non-disease-associated nucleic acid sequence.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a gene. In some embodiments, a common base sequencecomprises or is a sequence 100% complementary to a gene. In someembodiments, a common base sequence comprises or is a sequencecomplementary to a characteristic sequence element of a gene, whichcharacteristic sequences differentiate the gene from a similar sequencesharing homology with the gene. In some embodiments, a common basesequence comprises or is a sequence 100% complementary to acharacteristic sequence element of a gene, which characteristicsequences differentiate the gene from a similar sequence sharinghomology with the gene. In some embodiments, a common base sequencecomprises or is a sequence complementary to characteristic sequenceelement of a target gene, which characteristic sequences comprises amutation that is not found in other copies of the gene, e.g., thewild-type copy of the gene, another mutant copy the gene, etc. In someembodiments, a common base sequence comprises or is a sequence 100%complementary to characteristic sequence element of a target gene, whichcharacteristic sequences comprises a mutation that is not found in othercopies of the gene, e.g., the wild-type copy of the gene, another mutantcopy the gene, etc.

In some embodiments, a common base sequence comprises or is a sequencecomplementary to a sequence comprising an SNP. In some embodiments, acommon base sequence comprises or is a sequence complementary to asequence comprising an SNP, and the common base sequence is 100%complementary to the SNP that is associated with a disease. For example,in some embodiments, a common base sequence is 100% complementary to anSNP associated with a Huntington's disease-associated (or -causing)allele. In some embodiments, a common base sequence is that of WV-1092,which is 100% complementary to the disease-causing allele in manyHuntington's disease patients at rs362307. In some embodiments, an SNPis rs362307. In some embodiments, an SNP is rs7685686. In someembodiments, an SNP is rs362268. In some embodiments, an SNP isrs362306. In some embodiments, other example SNP site may be any of theHuntingtin site disclosed in the present disclosure.

In some embodiments, a common base sequence comprises a sequence foundin GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 16). In some embodiments, a commonbase sequence comprises a sequence found in GCCTCAGTCTGCTTCGCACC (SEQ IDNO: 16), wherein the sequence found in GCCTCAGTCTGCTTCGCACC (SEQ ID NO:16) comprises at least 15 nucleotides. In some embodiments, a commonbase sequence is GCCTCAGTCTGCTTCGCACC (SEQ ID NO: 16).

In some embodiments, a common base sequence comprises a sequence foundin GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 17). In some embodiments, a commonbase sequence comprises a sequence found in GAGCAGCTGCAACCTGGCAA (SEQ IDNO: 17), wherein the sequence found in GAGCAGCTGCAACCTGGCAA (SEQ ID NO:17) comprises at least 15 nucleotides. In some embodiments, a commonbase sequence is GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 17). In someembodiments, a common base sequence is GGGCACAAGGGCACAGACTT (SEQ ID NO:18). In some embodiments, a common base sequence is GAGCAGCTGCAACCTGGCAA(SEQ ID NO: 17). In some embodiments, a common base sequence isGCACAAGGGCACAGACTTCC (SEQ ID NO: 19). In some embodiments, a common basesequence is CACAAGGGCACAGACTTCCA (SEQ ID NO: 20). In some embodiments, acommon base sequence is ACAAGGGCACAGACTTCCAA (SEQ ID NO: 21). In someembodiments, a common base sequence is CAAGGGCACAGACTTCCAAA (SEQ ID NO:22). In some embodiments, a common base sequence comprises a sequencefound in GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 17). In some embodiments, acommon base sequence comprises a sequence found in GAGCAGCTGCAACCTGGCAA(SEQ ID NO: 17), wherein the sequence found in GAGCAGCTGCAACCTGGCAA (SEQID NO: 17) comprises at least 15 nucleotides. In some embodiments, acommon base sequence is GAGCAGCTGCAACCTGGCAA (SEQ ID NO: 17). In someembodiments, a common base sequence is GAGCAGCTGCAACCTGGCAA (SEQ ID NO:17). In some embodiments, a common base sequence is AGCAGCTGCAACCTGGCAAC(SEQ ID NO: 23). In some embodiments, a common base sequence isGCAGCTGCAACCTGGCAACA (SEQ ID NO: 24). In some embodiments, a common basesequence is CAGCTGCAACCTGGCAACAA (SEQ ID NO: 25). In some embodiments, acommon base sequence is AGCTGCAACCTGGCAACAAC (SEQ ID NO: 26). In someembodiments, a common base sequence is GCTGCAACCTGGCAACAACC (SEQ ID NO:27). In some embodiments, a common base sequence comprises a sequencefound in GGGCCAACAGCCAGCCTGCA (SEQ ID NO: 28). In some embodiments, acommon base sequence comprises a sequence found in GGGCCAACAGCCAGCCTGCA(SEQ ID NO: 28), wherein the sequence found in GGGCCAACAGCCAGCCTGCA (SEQID NO: 28) comprises at least 15 nucleotides. In some embodiments, acommon base sequence is GGGCCAACAGCCAGCCTGCA (SEQ ID NO: 28). In someembodiments, a common base sequence is GGGCCAACAGCCAGCCTGCA (SEQ ID NO:28). In some embodiments, a common base sequence is GGCCAACAGCCAGCCTGCAG(SEQ ID NO: 29). In some embodiments, a common base sequence isGCCAACAGCCAGCCTGCAGG (SEQ ID NO: 30). In some embodiments, a common basesequence is CCAACAGCCAGCCTGCAGGA (SEQ ID NO: 31). In some embodiments, acommon base sequence is CAACAGCCAGCCTGCAGGAG (SEQ ID NO: 32). In someembodiments, a common base sequence is AACAGCCAGCCTGCAGGAGG (SEQ ID NO:33). In some embodiments, a common base sequence comprises a sequencefound in ATTAATAAATTGTCATCACC (SEQ ID NO: 34). In some embodiments, acommon base sequence comprises a sequence found in ATTAATAAATTGTCATCACC(SEQ ID NO: 34), wherein the sequence found in ATTAATAAATTGTCATCACC (SEQID NO: 34) comprises at least 15 nucleotides. In some embodiments, acommon base sequence is ATTAATAAATTGTCATCACC (SEQ ID NO: 34). In someembodiments, a common base sequence is ATTAATAAATTGTCATCACC (SEQ ID NO:34).

In some embodiments, a chiral internucleotidic linkage has the structureof formula I. In some embodiments, a chiral internucleotidic linkage isphosphorothioate. In some embodiments, each chiral internucleotidiclinkage in a single oligonucleotide of a provided compositionindependently has the structure of formula I. In some embodiments, eachchiral internucleotidic linkage in a single oligonucleotide of aprovided composition is a phosphorothioate.

In some embodiments, oligonucleotides of the present disclosure compriseone or more modified sugar moieties. In some embodiments,oligonucleotides of the present disclosure comprise one or more modifiedbase moieties. As known by a person of ordinary skill in the art anddescribed in the disclosure, various modifications can be introduced toa sugar and/or moiety. For example, in some embodiments, a modificationis a modification described in U.S. Pat. No. 9,006,198, WO2014/012081and WO/2015/107425, the sugar and base modifications of each of whichare incorporated herein by reference.

In some embodiments, a sugar modification is a 2′-modification. Commonlyused 2′-modifications include but are not limited to 2′-OR¹, wherein R¹is not hydrogen. In some embodiments, a modification is 2′-OR, wherein Ris optionally substituted aliphatic. In some embodiments, a modificationis 2′-OMe. In some embodiments, a modification is 2′-MOE. In someembodiments, the present disclosure demonstrates that inclusion and/orlocation of particular chirally pure internucleotidic linkages canprovide stability improvements comparable to or better than thoseachieved through use of modified backbone linkages, bases, and/orsugars. In some embodiments, a provided single oligonucleotide of aprovided composition has no modifications on the sugars. In someembodiments, a provided single oligonucleotide of a provided compositionhas no modifications on 2′-positions of the sugars (i.e., the two groupsat the 2′-position are either —H/—H or —H/—OH). In some embodiments, aprovided single oligonucleotide of a provided composition does not haveany 2′-MOE modifications.

In some embodiments, a 2′-modification is —O-L- or -L- which connectsthe 2′-carbon of a sugar moiety to another carbon of a sugar moiety. Insome embodiments, a 2′-modification is —O-L- or -L- which connects the2′-carbon of a sugar moiety to the 4′-carbon of a sugar moiety. In someembodiments, a 2′-modification is S-cEt. In some embodiments, a modifiedsugar moiety is an LNA moiety.

In some embodiments, a 2′-modification is —F. In some embodiments, a2′-modification is FANA. In some embodiments, a 2′-modification is FRNA.

In some embodiments, a sugar modification is a 5′-modification, e.g.,R-5′-Me, S-5′-Me, etc.

In some embodiments, a sugar modification changes the size of the sugarring. In some embodiments, a sugar modification is the sugar moiety inFHNA.

In some embodiments, a sugar modification replaces a sugar moiety withanother cyclic or acyclic moiety. Example such moieties are widely knownin the art, including but not limited to those used in morpholio(optionally with its phosphorodiamidate linkage), glycol nucleic acids,etc.

In some embodiments, a single oligonucleotide in a provided compositionhas at least about 25% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 30% of its internucleotidiclinkages in Sp configuration. In some embodiments, a singleoligonucleotide in a provided composition has at least about 35% of itsinternucleotidic linkages in Sp configuration. In some embodiments, asingle oligonucleotide in a provided composition has at least about 40%of its internucleotidic linkages in Sp configuration. In someembodiments, a single oligonucleotide in a provided composition has atleast about 45% of its internucleotidic linkages in Sp configuration. Insome embodiments, a single oligonucleotide in a provided composition hasat least about 50% of its internucleotidic linkages in Sp configuration.In some embodiments, a single oligonucleotide in a provided compositionhas at least about 55% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 60% of its internucleotidiclinkages in Sp configuration. In some embodiments, a singleoligonucleotide in a provided composition has at least about 65% of itsinternucleotidic linkages in Sp configuration. In some embodiments, asingle oligonucleotide in a provided composition has at least about 70%of its internucleotidic linkages in Sp configuration. In someembodiments, a single oligonucleotide in a provided composition has atleast about 75% of its internucleotidic linkages in Sp configuration. Insome embodiments, a single oligonucleotide in a provided composition hasat least about 80% of its internucleotidic linkages in Sp configuration.In some embodiments, a single oligonucleotide in a provided compositionhas at least about 85% of its internucleotidic linkages in Spconfiguration. In some embodiments, a single oligonucleotide in aprovided composition has at least about 90% of its internucleotidiclinkages in Sp configuration.

In some embodiments, oligonucleotides in a provided composition is notan oligonucleotide selected from:TkTk^(m)C_(k)AGT^(m)CATGA^(m)CT_(k)T^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 35),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methylcytosine modified nucleoside, and allinternucleoside linkages are phosphorothioates (PS) with stereochemistrypatterns selected from RSSSRSRRRS, RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR,RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS,SSSSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR,RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS,RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS,SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR and SSSSSSSSSS.

In some embodiments, a single oligonucleotide in a provided compositionis not an oligonucleotide selected from:TkTk^(m)C_(k)AGT^(m)CATGA^(m)CTT_(k) ^(m)C_(k) ^(m)C_(k) (SEQ ID NO:36), wherein each nucleoside followed by a subscript ‘k’ indicates a(S)-cEt modification, R is Rp phosphorothioate linkage, S is Spphosphorothioate linkage, each ^(m)C is a 5-methylcytosine modifiednucleoside and all core internucleoside linkages are phosphorothioates(PS) with stereochemistry patterns selected from: RSSSRSRRRS,RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR,SRSSSRSSSS, SSRRSSRSRS, SSSSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS,SRRSRRRRRR, RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR,RRSSRSRRRR, RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR,RSRSRSSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRRand SSSSSSSSSS.

Chirally Controlled Oligonucleotides and Chirally ControlledOligonucleotide Compositions

The present disclosure provides chirally controlled oligonucleotides,and chirally controlled oligonucleotide compositions which are of highcrude purity and of high diastereomeric purity. In some embodiments, thepresent disclosure provides chirally controlled oligonucleotides, andchirally controlled oligonucleotide compositions which are of high crudepurity. In some embodiments, the present disclosure provides chirallycontrolled oligonucleotides, and chirally controlled oligonucleotidecompositions which are of high diastereomeric purity.

In some embodiments, a chirally controlled oligonucleotide compositionis a substantially pure preparation of an oligonucleotide type in thatoligonucleotides in the composition that are not of the oligonucleotidetype are impurities form the preparation process of said oligonucleotidetype, in some case, after certain purification procedures.

In some embodiments, the present disclosure provides oligonucleotidescomprising one or more diastereomerically pure internucleotidic linkageswith respect to the chiral linkage phosphorus. In some embodiments, thepresent disclosure provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I. In some embodiments, the present disclosure providesoligonucleotides comprising one or more diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus,and one or more phosphate diester linkages. In some embodiments, thepresent disclosure provides oligonucleotides comprising one or morediastereomerically pure internucleotidic linkages having the structureof formula I, and one or more phosphate diester linkages. In someembodiments, the present disclosure provides oligonucleotides comprisingone or more diastereomerically pure internucleotidic linkages having thestructure of formula I-c, and one or more phosphate diester linkages. Insome embodiments, such oligonucleotides are prepared by usingstereoselective oligonucleotide synthesis, as described in thisapplication, to form pre-designed diastereomerically pureinternucleotidic linkages with respect to the chiral linkage phosphorus.For instance, in one example oligonucleotide of (Rp/Sp, Rp/Sp, Rp/Sp,Rp, Rp, Sp, Sp, Sp, Sp, Sp Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGs1Cs1As1CsC] (SEQ ID NO: 37), thefirst three internucleotidic linkages are constructed using traditionaloligonucleotide synthesis method, and the diastereomerically pureinternucleotidic linkages are constructed with stereochemical control asdescribed in this application. Example internucleotidic linkages,including those having structures of formula I, are further describedbelow.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In certain embodiments, the present disclosure provides achirally controlled oligonucleotide, wherein at least two individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another. In certain embodiments, thepresent disclosure provides a chirally controlled oligonucleotide,wherein at least two of the individual internucleotidic linkages withinthe oligonucleotide have different P-modifications relative to oneanother, and wherein the chirally controlled oligonucleotide comprisesat least one phosphate diester internucleotidic linkage. In certainembodiments, the present disclosure provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate diesterinternucleotidic linkage. In certain embodiments, the present disclosureprovides a chirally controlled oligonucleotide, wherein at least two ofthe individual internucleotidic linkages within the oligonucleotide havedifferent P-modifications relative to one another, and wherein thechirally controlled oligonucleotide comprises at least onephosphorothioate triester internucleotidic linkage. In certainembodiments, the present disclosure provides a chirally controlledoligonucleotide, wherein at least two of the individual internucleotidiclinkages within the oligonucleotide have different P-modificationsrelative to one another, and wherein the chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterinternucleotidic linkage.

In certain embodiments, a modified internucleotidic linkages has thestructure of formula I:

wherein each variable is as defined and described below. In someembodiments, a linkage of formula I is chiral. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising one or more modified internucleotidic linkages of formula I.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages of formula I, and wherein individualinternucleotidic linkages of formula I within the oligonucleotide havedifferent P-modifications relative to one another. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomprising one or more modified internucleotidic linkages of formula I,and wherein individual internucleotidic linkages of formula I within theoligonucleotide have different —X-L-R¹ relative to one another. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising one or more modified internucleotidiclinkages of formula I, and wherein individual internucleotidic linkagesof formula I within the oligonucleotide have different X relative to oneanother. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising one or more modifiedinternucleotidic linkages of formula I, and wherein individualinternucleotidic linkages of formula I within the oligonucleotide havedifferent -L-R¹ relative to one another. In some embodiments, a chirallycontrolled oligonucleotide is an oligonucleotide in a providedcomposition that is of the particular oligonucleotide type. In someembodiments, a chirally controlled oligonucleotide is an oligonucleotidein a provided composition that has the common base sequence and length,the common pattern of backbone linkages, and the common pattern ofbackbone chiral centers.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to oneanother. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry relative to one another, and wherein at least a portionof the structure of the chirally controlled oligonucleotide ischaracterized by a repeating pattern of alternating stereochemisty.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentP-modifications relative to one another, in that they have different Xatoms in their -XLR¹ moieties, and/or in that they have different Lgroups in their -XLR¹ moieties, and/or that they have different R¹ atomsin their -XLR¹ moieties.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide, wherein at least two of the individualinternucleotidic linkages within the oligonucleotide have differentstereochemistry and/or different P-modifications relative to one anotherand the oligonucleotide has a structure represented by the followingformula:

[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny]

wherein:each R^(B) independently represents a block of nucleotide units havingthe R configuration at the linkage phosphorus;each S^(B) independently represents a block of nucleotide units havingthe S configuration at the linkage phosphorus;each of n1-ny is zero or an integer, with the requirement that at leastone odd n and at least one even n must be non-zero so that theoligonucleotide includes at least two individual internucleotidiclinkages with different stereochemistry relative to one another; andwherein the sum of n1-ny is between 2 and 200, and in some embodimentsis between a lower limit selected from the group consisting of 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or more and an upper limit selected from the group consisting of5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upperlimit being larger than the lower limit.

In some such embodiments, each n has the same value; in someembodiments, each even n has the same value as each other even n; insome embodiments, each odd n has the same value each other odd n; insome embodiments, at least two even ns have different values from oneanother; in some embodiments, at least two odd ns have different valuesfrom one another.

In some embodiments, at least two adjacent ns are equal to one another,so that a provided oligonucleotide includes adjacent blocks of Sstereochemistry linkages and R stereochemistry linkages of equallengths. In some embodiments, provided oligonucleotides includerepeating blocks of S and R stereochemistry linkages of equal lengths.In some embodiments, provided oligonucleotides include repeating blocksof S and R stereochemistry linkages, where at least two such blocks areof different lengths from one another; in some such embodiments each Sstereochemistry block is of the same length, and is of a differentlength from each R stereochemistry length, which may optionally be ofthe same length as one another.

In some embodiments, at least two skip-adjacent ns are equal to oneanother, so that a provided oligonucleotide includes at least two blocksof linkages of a first steroechemistry that are equal in length to oneanother and are separated by a block of linkages of the otherstereochemistry, which separating block may be of the same length or adifferent length from the blocks of first steroechemistry.

In some embodiments, ns associated with linkage blocks at the ends of aprovided oligonucleotide are of the same length. In some embodiments,provided oligonucleotides have terminal blocks of the same linkagestereochemistry. In some such embodiments, the terminal blocks areseparated from one another by a middle block of the other linkagestereochemistry.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoblockmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoskipmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a stereoaltmer.In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a gapmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is of any of theabove described patterns and further comprises patterns ofP-modifications. For instance, in some embodiments, a providedoligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . .S^(B)nxR^(B)ny] and is a stereoskipmer and P-modification skipmer. Insome embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] and is astereoblockmer and P-modification altmer. In some embodiments, aprovided oligonucleotide of formula [S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . .S^(B)nxR^(B)ny] and is a stereoaltmer and P-modification blockmer.

In some embodiments, a provided oligonucleotide of formula[S^(B)n1R^(B)n2S^(B)n3R^(B)n4 . . . S^(B)nxR^(B)ny] is a chirallycontrolled oligonucleotide comprising one or more modifiedinternuceotidic linkages independently having the structure of formulaI:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   W is O, S or Se;-   each of X, Y and Z is independently —O—, —S—, —N(-L-R¹)—, or L;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted group selected from C₁-C₆ alkylene, C₁-C₆ alkenylene,    —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—,    —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—, and    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted group selected from C₁-C₆    alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,    —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,    —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,    —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—,    —C(O)S—, —OC(O)—, and —C(O)O—-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, and    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, carbocyclyl, aryl, heteroaryl, and    heterocyclyl; and-   each

independently represents a connection to a nucleoside.

In some embodiments, L is a covalent bond or an optionally substituted,linear or branched C₁-C₁₀ alkylene, wherein one or more methylene unitsof L are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—;

-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl; and-   each

independently represents a connection to a nucleoside.

In some embodiments, a chirally controlled oligonucleotide comprises oneor more modified internucleotidic phosphorus linkages. In someembodiments, a chirally controlled oligonucleotide comprises, e.g., aphosphorothioate or a phosphorothioate triester linkage. In someembodiments, a chirally controlled oligonucleotide comprises aphosphorothioate triester linkage. In some embodiments, a chirallycontrolled oligonucleotide comprises at least two phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least three phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least four phosphorothioate triester linkages. In someembodiments, a chirally controlled oligonucleotide comprises at leastfive phosphorothioate triester linkages. Example such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a chirally controlled oligonucleotide comprisesdifferent internucleotidic phosphorus linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least one modifiedinternucleotidic linkage. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast two phosphorothioate triester linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages. In some embodiments, a chirally controlledoligonucleotide comprises at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages. In some embodiments, a chirally controlled oligonucleotidecomprises at least one phosphate diester internucleotidic linkage and atleast five phosphorothioate triester linkages. Example such modifiedinternucleotidic phosphorus linkages are described further herein.

In some embodiments, a phosphorothioate triester linkage comprises achiral auxiliary, which, for example, is used to control thestereoselectivity of a reaction. In some embodiments, a phosphorothioatetriester linkage does not comprise a chiral auxiliary. In someembodiments, a phosphorothioate triester linkage is intentionallymaintained until and/or during the administration to a subject.

In some embodiments, a chirally controlled oligonucleotide is linked toa solid support. In some embodiments, a chirally controlledoligonucleotide is cleaved from a solid support.

In some embodiments, a chirally controlled oligonucleotide comprises atleast one phosphate diester internucleotidic linkage and at least twoconsecutive modified internucleotidic linkages. In some embodiments, achirally controlled oligonucleotide comprises at least one phosphatediester internucleotidic linkage and at least two consecutivephosphorothioate triester internucleotidic linkages.

In some embodiments, a chirally controlled oligonucleotide is ablockmer. In some embodiments, a chirally controlled oligonucleotide isa stereoblockmer. In some embodiments, a chirally controlledoligonucleotide is a P-modification blockmer. In some embodiments, achirally controlled oligonucleotide is a linkage blockmer.

In some embodiments, a chirally controlled oligonucleotide is an altmer.In some embodiments, a chirally controlled oligonucleotide is astereoaltmer. In some embodiments, a chirally controlled oligonucleotideis a P-modification altmer. In some embodiments, a chirally controlledoligonucleotide is a linkage altmer.

In some embodiments, a chirally controlled oligonucleotide is a unimer.In some embodiments, a chirally controlled oligonucleotide is astereounimer. In some embodiments, a chirally controlled oligonucleotideis a P-modification unimer. In some embodiments, a chirally controlledoligonucleotide is a linkage unimer.

In some embodiments, a chirally controlled oligonucleotide is a gapmer.

In some embodiments, a chirally controlled oligonucleotide is a skipmer.

In some embodiments, the present disclosure provides oligonucleotidescomprising one or more modified internucleotidic linkages independentlyhaving the structure of formula I:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   W is O, S or Se;-   each of X, Y and Z is independently —O—, —S—, —N(-L-R¹)—, or L;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted group selected from C₁-C₆ alkylene, C₁-C₆ alkenylene,    —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—,    —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—, and    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted group selected from C₁-C₆    alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,    —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,    —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,    —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—,    —C(O)S—, —OC(O)—, and —C(O)O—-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, and    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, carbocyclyl, aryl, heteroaryl, and    heterocyclyl; and-   each

independently represents a connection to a nucleoside.

In some embodiments, L is a covalent bond or an optionally substituted,linear or branched C₁-C₁₀ alkylene, wherein one or more methylene unitsof L are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—;

-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl; and-   each

independently represents a connection to a nucleoside.

In some embodiments, P* is an asymmetric phosphorus atom and is eitherRp or Sp. In some embodiments, P* is Rp. In other embodiments, P* is Sp.In some embodiments, an oligonucleotide comprises one or moreinternucleotidic linkages of formula I wherein each P* is independentlyRp or Sp. In some embodiments, an oligonucleotide comprises one or moreinternucleotidic linkages of formula I wherein each P* is Rp. In someembodiments, an oligonucleotide comprises one or more internucleotidiclinkages of formula I wherein each P* is Sp. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein P* is Rp. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein P*is Sp. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein P* is Rp, and at least oneinternucleotidic linkage of formula I wherein P* is Sp.

In some embodiments, W is O, S, or Se. In some embodiments, W is O. Insome embodiments, W is S. In some embodiments, W is Se. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein W is O. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein W is S. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein Wis Se.

In some embodiments, each R is independently hydrogen, or an optionallysubstituted group selected from C₁-C₆ aliphatic, phenyl, carbocyclyl,aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is hydrogen. In some embodiments, R is anoptionally substituted group selected from C₁-C₆ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R is an optionally substituted C₁-C₆ alkyl. In someembodiments, R is optionally substituted, linear or branched hexyl. Insome embodiments, R is optionally substituted, linear or branchedpentyl. In some embodiments, R is optionally substituted, linear orbranched butyl. In some embodiments, R is optionally substituted, linearor branched propyl. In some embodiments, R is optionally substitutedethyl. In some embodiments, R is optionally substituted methyl.

In some embodiments, R is optionally substituted phenyl. In someembodiments, R is substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, R is optionally substituted carbocyclyl. In someembodiments, R is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R is optionally substituted monocyclic carbocyclyl. In someembodiments, R is optionally substituted cycloheptyl. In someembodiments, R is optionally substituted cyclohexyl. In someembodiments, R is optionally substituted cyclopentyl. In someembodiments, R is optionally substituted cyclobutyl. In someembodiments, R is an optionally substituted cyclopropyl. In someembodiments, R is optionally substituted bicyclic carbocyclyl.

In some embodiments, R is an optionally substituted aryl. In someembodiments, R is an optionally substituted bicyclic aryl ring.

In some embodiments, R is an optionally substituted heteroaryl. In someembodiments, R is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R is a substituted 5-6membered monocyclic heteroaryl ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, sulfur, or oxygen.

In some embodiments, R is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R is an optionallysubstituted 6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Example R groupsinclude optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R is a 6-membered heteroaryl ring having 1-3nitrogen atoms. In other embodiments, R is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Example Rgroups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R is an optionally substituted 5,6-fusedheteroaryl ring having 1 heteroatom independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted indolyl. In some embodiments, R is an optionally substitutedazabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionallysubstituted 5,6-fused heteroaryl ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted azaindolyl. In some embodiments, R is anoptionally substituted benzimidazolyl. In some embodiments, R is anoptionally substituted benzothiazolyl. In some embodiments, R is anoptionally substituted benzoxazolyl. In some embodiments, R is anoptionally substituted indazolyl. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R is an optionally substituted quinolinyl.In some embodiments, R is an optionally substituted isoquinolinyl.According to one aspect, R is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R is a quinazoline ora quinoxaline.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R is an optionally substituted heterocyclyl. Insome embodiments, R is an optionally substituted 6 membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atom.

In certain embodiments, R is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, Ris an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R is an optionally substituted indolinyl. In someembodiments, R is an optionally substituted isoindolinyl. In someembodiments, R is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

In some embodiments, each R′ is independently —R, —C(O)R, —CO₂R, or—SO₂R, or:

-   -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring.

In some embodiments, R′ is —R, —C(O)R, —CO₂R, or —SO₂R, wherein R is asdefined above and described herein.

In some embodiments, R′ is —R, wherein R is as defined and describedabove and herein. In some embodiments, R′ is hydrogen.

In some embodiments, R′ is —C(O)R, wherein R is as defined above anddescribed herein. In some embodiments, R′ is —CO₂R, wherein R is asdefined above and described herein. In some embodiments, R′ is —SO₂R,wherein R is as defined above and described herein.

In some embodiments, two R′ on the same nitrogen are taken together withtheir intervening atoms to form an optionally substituted heterocyclicor heteroaryl ring. In some embodiments, two R′ on the same carbon aretaken together with their intervening atoms to form an optionallysubstituted aryl, carbocyclic, heterocyclic, or heteroaryl ring.

In some embodiments, -Cy- is an optionally substituted bivalent ringselected from phenylene, carbocyclylene, arylene, heteroarylene, orheterocyclylene.

In some embodiments, -Cy- is optionally substituted phenylene. In someembodiments, -Cy- is optionally substituted carbocyclylene. In someembodiments, -Cy- is optionally substituted arylene. In someembodiments, -Cy- is optionally substituted heteroarylene. In someembodiments, -Cy- is optionally substituted heterocyclylene.

In some embodiments, each of X, Y and Z is independently —O—, —S—,—N(-L-R¹)—, or L, wherein each of L and R¹ is independently as definedabove and described below.

In some embodiments, X is —O—. In some embodiments, X is —S—. In someembodiments, X is —O— or —S—. In some embodiments, an oligonucleotidecomprises at least one internucleotidic linkage of formula I wherein Xis —O—. In some embodiments, an oligonucleotide comprises at least oneinternucleotidic linkage of formula I wherein X is —S—. In someembodiments, an oligonucleotide comprises at least one internucleotidiclinkage of formula I wherein X is —O—, and at least one internucleotidiclinkage of formula I wherein X is —S—. In some embodiments, anoligonucleotide comprises at least one internucleotidic linkage offormula I wherein X is —O—, and at least one internucleotidic linkage offormula I wherein X is —S—, and at least one internucleotidic linkage offormula I wherein L is an optionally substituted, linear or branchedC₁-C₁₀ alkylene, wherein one or more methylene units of L are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—.

In some embodiments, X is —N(-L-R¹)—. In some embodiments, X is —N(R¹)—.

In some embodiments, X is —N(R′)—. In some embodiments, X is —N(R)—. Insome embodiments, X is —NH—.

In some embodiments, X is L. In some embodiments, X is a covalent bond.In some embodiments, X is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, X is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, X is methylene.

In some embodiments, Y is —O—. In some embodiments, Y is —S—.

In some embodiments, Y is —N(-L-R¹)—. In some embodiments, Y is —N(R¹)—.In some embodiments, Y is —N(R′)—. In some embodiments, Y is —N(R)—. Insome embodiments, Y is —NH—.

In some embodiments, Y is L. In some embodiments, Y is a covalent bond.In some embodiments, Y is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Y is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Y is methylene.

In some embodiments, Z is —O—. In some embodiments, Z is —S—.

In some embodiments, Z is —N(-L-R¹)—. In some embodiments, Z is —N(R¹)—.In some embodiments, Z is —N(R′)—. In some embodiments, Z is —N(R)—. Insome embodiments, Z is —NH—.

In some embodiments, Z is L. In some embodiments, Z is a covalent bond.In some embodiments, Z is or an optionally substituted, linear orbranched C₁-C₁₀ alkylene, wherein one or more methylene units of L areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In someembodiments, Z is an optionally substituted C₁-C₁₀ alkylene or C₁-C₁₀alkenylene. In some embodiments, Z is methylene.

In some embodiments, L is a covalent bond or an optionally substituted,linear or branched C₁-C₁₀ alkylene, wherein one or more methylene unitsof L are optionally and independently replaced by an optionallysubstituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or—C(O)O—.

In some embodiments, L is a covalent bond. In some embodiments, L is anoptionally substituted, linear or branched C₁-C₁₀ alkylene, wherein oneor more methylene units of L are optionally and independently replacedby an optionally substituted C₁-C₆ alkylene, C₁—C₆ alkenylene, —C≡C—,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—.

In some embodiments, L has the structure of -L¹-V— wherein:

L¹ is an optionally substituted group selected from

C₁-C₆ alkylene, C₁-C₆ alkenylene, carbocyclylene, arylene, C₁-C₆heteroalkylene, heterocyclylene, and heteroarylene;V is selected from —O—, —S—, —NR′—, C(R′)₂, —S—S—, —B—S—S—C—,

or an optionally substituted group selected from C₁-C₆ alkylene,arylene, C₁-C₆ heteroalkylene, heterocyclylene, and heteroarylene;

A is ═O, ═S, ═NR′, or ═C(R′)₂;

each of B and C is independently —O—, —S—, —NR′—, —C(R′)₂—, or anoptionally substituted group selected from C₁-C₆ alkylene,carbocyclylene, arylene, heterocyclylene, or heteroarylene; and each R′is independently as defined above and described herein.

In some embodiments, L¹ is,

In some embodiments, L¹ is

wherein Ring Cy′ is an optionally substituted arylene, carbocyclylene,heteroarylene, or heterocyclylene. In some embodiments, L¹ is optionallysubstituted

In some embodiments, L¹ is

In some embodiments, L¹ is connected to X. In some embodiments, L¹ is anoptionally substituted group selected

and the sulfur atom is connect to V. In some embodiments, L¹ is anoptionally substituted group selected from

and the carbon atom is connect to X.

In some embodiments, L has the structure of:

wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;

is a single or double bond;the two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, carbocyclic,heteroaryl or heterocyclic ring; and each R′ is independently as definedabove and described herein.

In some embodiments, L has the structure of:

wherein:

G is —O—, —S—, or —NR′;

is a single or double bond; andthe two R^(L1) are taken together with the two carbon atoms to whichthey are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   is a single or double bond;-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond;-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   is a single or double bond;-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond;-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring; and each R′ is    independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   R′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   E is —O—, —S—, —NR′— or —C(R′)₂—;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—,    ═C(CO₂—(C₁-C₆ aliphatic))-, or ═C(CF₃)—; and-   R′ is as defined above and described herein.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein the phenyl ring is optionally substituted. In some embodiments,the phenyl ring is not substituted. In some embodiments, the phenyl ringis substituted.

In some embodiments, L has the structure of:

wherein:

-   is a single or double bond; and-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

wherein:

-   G is —O—, —S—, or —NR′;-   is a single or double bond; and-   the two R^(L1) are taken together with the two carbon atoms to which    they are bound to form an optionally substituted aryl, C₃-C₁₀    carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, E is —O—, —S—, —NR′— or —C(R′)₂—, wherein each R′independently as defined above and described herein. In someembodiments, E is —O—, —S—, or —NR′—. In some embodiments, E is —O—,—S—, or —NH—. In some embodiments, E is —O—. In some embodiments, E is—S—. In some embodiments, E is —NH—.

In some embodiments, G is —O—, —S—, or —NR′, wherein each R′independently as defined above and described herein. In someembodiments, G is —O—, —S—, or —NH—. In some embodiments, G is —O—. Insome embodiments, G is —S—. In some embodiments, G is —NH—.

In some embodiments, L is -L³-G-, wherein:

-   L³ is an optionally substituted C₁-C₅ alkylene or alkenylene,    wherein one or more methylene units are optionally and independently    replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—,    —S(O)₂—, or

and

-   wherein each of G, R′ and Ring Cy′ is independently as defined above    and described herein.

In some embodiments, L is -L³-S—, wherein L³ is as defined above anddescribed herein. In some embodiments, L is -L³-O—, wherein L³ is asdefined above and described herein. In some embodiments, L is-L³-N(R′)—, wherein each of L³ and R′ is independently as defined aboveand described herein. In some embodiments, L is -L³-NH—, wherein each ofL³ and R′ is independently as defined above and described herein.

In some embodiments, L³ is an optionally substituted C₅ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Ring Cy′ is independently as defined above anddescribed herein. In some embodiments, L³ is an optionally substitutedC₅ alkylene. In some embodiments, -L³-G- is

In some embodiments, L³ is an optionally substituted C₄ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

In some embodiments, L³ is an optionally substituted C₃ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments, L³ is an optionally substituted C₂ alkylene oralkenylene, wherein one or more methylene units are optionally andindependently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—S(O)—, —S(O)₂—, or

and each of R′ and Cy′ is independently as defined above and describedherein.

In some embodiments, -L³-G- is

wherein each of G and Cy′ is independently as defined above anddescribed herein. In some embodiments, L is

In some embodiments, L is -L⁴-G-, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; and G is as defined above and describedherein. In some embodiments, L is -L⁴-G-, wherein L⁴ is an optionallysubstituted C₁-C₂ alkylene; G is as defined above and described herein;and G is connected to R¹. In some embodiments, L is -L⁴-G-, wherein L⁴is an optionally substituted methylene; G is as defined above anddescribed herein; and G is connected to R¹. In some embodiments, L is-L⁴-G-, wherein L⁴ is methylene; G is as defined above and describedherein; and G is connected to R¹. In some embodiments, L is -L⁴-G-,wherein L⁴ is an optionally substituted —(CH₂)₂—; G is as defined aboveand described herein; and G is connected to R¹. In some embodiments, Lis -L⁴-G-, wherein L⁴ is —(CH₂)₂—; G is as defined above and describedherein; and G is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein G is as defined above and described herein, and G is connectedto R¹. In some embodiments, L is

wherein the sulfur atom is connected to R¹. In some embodiments, L is

wherein the oxygen atom is connected to R¹.

In some embodiments, L is

wherein G is as defined above and described herein.

In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3)is an optionally substituted, linear or branched, C₁-C₉ alkylene,wherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each of R′ and -Cy- isindependently as defined above and described herein. In someembodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is anoptionally substituted C₁-C₆ alkylene. In some embodiments, L is—S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is an optionallysubstituted C₁-C₆ alkenylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkylene wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkenylene,arylene, or heteroarylene. In some embodiments, In some embodiments,R^(L3) is an optionally substituted —S—(C₁-C₆ alkenylene)-, —S—(C₁-C₆alkylene)-, —S—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)-,—S—CO-arylene-(C₁-C₆ alkylene)-, or —S—CO—(C₁-C₆alkylene)-arylene-(C₁-C₆ alkylene)-.

In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments,

In some embodiments, the sulfur atom in the L embodiments describedabove and herein is connected to X. In some embodiments, the sulfur atomin the L embodiments described above and herein is connected to R¹.

In some embodiments, R¹ is halogen, R, or an optionally substitutedC₁-C₅₀ aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein. In someembodiments, R¹ is halogen, R, or an optionally substituted C₁-C₁₀aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is halogen.In some embodiments, R¹ is —F. In some embodiments, R¹ is —Cl. In someembodiments, R¹ is —Br. In some embodiments, R¹ is —I.

In some embodiments, R¹ is R wherein R is as defined above and describedherein.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is anoptionally substituted group selected from C₁-C₅₀ aliphatic, phenyl,carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphatic.In some embodiments, R¹ is an optionally substituted C₁-C₆ aliphatic. Insome embodiments, R¹ is an optionally substituted C₁-C₆ alkyl. In someembodiments, R¹ is optionally substituted, linear or branched hexyl. Insome embodiments, R¹ is optionally substituted, linear or branchedpentyl. In some embodiments, R¹ is optionally substituted, linear orbranched butyl. In some embodiments, R¹ is optionally substituted,linear or branched propyl. In some embodiments, R¹ is optionallysubstituted ethyl. In some embodiments, R¹ is optionally substitutedmethyl.

In some embodiments, R¹ is optionally substituted phenyl. In someembodiments, R¹ is substituted phenyl. In some embodiments, R¹ isphenyl.

In some embodiments, R¹ is optionally substituted carbocyclyl. In someembodiments, R¹ is optionally substituted C₃-C₁₀ carbocyclyl. In someembodiments, R¹ is optionally substituted monocyclic carbocyclyl. Insome embodiments, R¹ is optionally substituted cycloheptyl. In someembodiments, R¹ is optionally substituted cyclohexyl. In someembodiments, R¹ is optionally substituted cyclopentyl. In someembodiments, R¹ is optionally substituted cyclobutyl. In someembodiments, R¹ is an optionally substituted cyclopropyl. In someembodiments, R¹ is optionally substituted bicyclic carbocyclyl.

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ polycyclichydrocarbon. In some embodiments, R¹ is an optionally substituted C₁-C₅₀polycyclic hydrocarbon wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein eachvariable is independently as defined above and described herein. In someembodiments, R¹ is optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is optionally substituted

In some embodiments, R¹ is an optionally substituted C₁-C₅₀ aliphaticcomprising one or more optionally substituted polycyclic hydrocarbonmoieties. In some embodiments, R¹ is an optionally substituted C₁-C₅₀aliphatic comprising one or more optionally substituted polycyclichydrocarbon moieties, wherein one or more methylene units are optionallyand independently replaced by an optionally substituted C₁-C₆ alkylene,C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,—N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—,—SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable isindependently as defined above and described herein. In someembodiments, R¹ is an optionally substituted C₁-C₅₀ aliphatic comprisingone or more optionally substituted

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted aryl. In someembodiments, R¹ is an optionally substituted bicyclic aryl ring.

In some embodiments, R¹ is an optionally substituted heteroaryl. In someembodiments, R¹ is an optionally substituted 5-6 membered monocyclicheteroaryl ring having 1-3 heteroatoms independently selected fromnitrogen, sulfur, or oxygen. In some embodiments, R¹ is a substituted5-6 membered monocyclic heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an unsubstituted 5-6 membered monocyclic heteroarylring having 1-3 heteroatoms independently selected from nitrogen,sulfur, or oxygen.

In some embodiments, R¹ is an optionally substituted 5 memberedmonocyclic heteroaryl ring having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1 heteroatom selected from nitrogen,oxygen, or sulfur. In some embodiments, R¹ is selected from pyrrolyl,furanyl, or thienyl.

In some embodiments, R¹ is an optionally substituted 5-memberedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In certain embodiments, R¹ is an optionallysubstituted 5-membered heteroaryl ring having 1 nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Example R¹ groupsinclude optionally substituted pyrazolyl, imidazolyl, thiazolyl,isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R¹ is a 6-membered heteroaryl ring having 1-3nitrogen atoms. In other embodiments, R¹ is an optionally substituted6-membered heteroaryl ring having 1-2 nitrogen atoms. In someembodiments, R¹ is an optionally substituted 6-membered heteroaryl ringhaving 2 nitrogen atoms. In certain embodiments, R¹ is an optionallysubstituted 6-membered heteroaryl ring having 1 nitrogen. Example R¹groups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, orsulfur. In certain embodiments, R¹ is an optionally substituted5,6-fused heteroaryl ring having 1 heteroatom independently selectedfrom nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted indolyl. In some embodiments, R¹ is an optionallysubstituted azabicyclo[3.2.1]octanyl. In certain embodiments, R¹ is anoptionally substituted 5,6-fused heteroaryl ring having 2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted azaindolyl. In someembodiments, R¹ is an optionally substituted benzimidazolyl. In someembodiments, R¹ is an optionally substituted benzothiazolyl. In someembodiments, R¹ is an optionally substituted benzoxazolyl. In someembodiments, R¹ is an optionally substituted indazolyl. In certainembodiments, R¹ is an optionally substituted 5,6-fused heteroaryl ringhaving 3 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

In certain embodiments, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In otherembodiments, R¹ is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, orsulfur. In some embodiments, R¹ is an optionally substituted quinolinyl.In some embodiments, R¹ is an optionally substituted isoquinolinyl.According to one aspect, R¹ is an optionally substituted 6,6-fusedheteroaryl ring having 2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur. In some embodiments, R¹ is a quinazoline ora quinoxaline.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 3-7 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-2 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

In some embodiments, R¹ is an optionally substituted heterocyclyl. Insome embodiments, R¹ is an optionally substituted 6 membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In someembodiments, R¹ is an optionally substituted 6 membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In some embodiments, R¹ is anoptionally substituted 6 membered partially unsaturated heterocyclicring having 2 oxygen atoms.

In certain embodiments, R¹ is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹ isoxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl,dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R¹is an optionally substituted 5 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In certain embodiments, R¹ is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. In certainembodiments, R¹ is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R¹ is an optionally substituted 8-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-4heteroatoms independently selected from nitrogen, oxygen, or sulfur. Insome embodiments, R¹ is an optionally substituted indolinyl. In someembodiments, R¹ is an optionally substituted isoindolinyl. In someembodiments, R¹ is an optionally substituted 1, 2, 3,4-tetrahydroquinoline. In some embodiments, R¹ is an optionallysubstituted 1, 2, 3, 4-tetrahydroisoquinoline.

In some embodiments, R¹ is an optionally substituted C₁-C₁₀ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene,—C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—,—OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—,—C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently asdefined above and described herein. In some embodiments, R¹ is anoptionally substituted C₁-C₁₀ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionally-Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,—S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —OC(O)—, or —C(O)O—, wherein eachR′ is independently as defined above and described herein. In someembodiments, R¹ is an optionally substituted C₁-C₁₀ aliphatic whereinone or more methylene units are optionally and independently replaced byan optionally-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —OC(O)—, or—C(O)O—, wherein each R′ is independently as defined above and describedherein.

In some embodiments, R¹ is

In some embodiments, R¹ is CH₃—,

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)₂-moiety which is connected to L. Example such R¹ groups aredepicted below:

In some embodiments, R¹ comprises a terminal optionally substituted—(CH₂)-moiety which is connected to L. Example such R¹ groups aredepicted below:

In some embodiments, R¹ is —S—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and-Cy- is independently as defined above and described herein. In someembodiments, R¹ is —S—R^(L2), wherein the sulfur atom is connected withthe sulfur atom in L group.

In some embodiments, R¹ is —C(O)—R^(L2), wherein R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—,—N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,—N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and-Cy- is independently as defined above and described herein. In someembodiments, R¹ is —C(O)—R^(L2), wherein the carbonyl group is connectedwith G in L group. In some embodiments, R¹ is —C(O)—R^(L2), wherein thecarbonyl group is connected with the sulfur atom in L group.

In some embodiments, R^(L2) is optionally substituted C₁-C₉ aliphatic.In some embodiments, R^(L2) is optionally substituted C₁-C₉ alkyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkenyl. Insome embodiments, R^(L2) is optionally substituted C₁-C₉ alkynyl. Insome embodiments, R^(L2) is an optionally substituted C₁-C₉ aliphaticwherein one or more methylene units are optionally and independentlyreplaced by -Cy- or —C(O)—. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by -Cy-. In some embodiments,R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted heterocycylene. In some embodiments, R^(L2) is anoptionally substituted C₁-C₉ aliphatic wherein one or more methyleneunits are optionally and independently replaced by an optionallysubstituted arylene. In some embodiments, R^(L2) is an optionallysubstituted C₁-C₉ aliphatic wherein one or more methylene units areoptionally and independently replaced by an optionally substitutedheteroarylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein one or more methylene units are optionally andindependently replaced by an optionally substituted C₃-C₁₀carbocyclylene. In some embodiments, R^(L2) is an optionally substitutedC₁-C₉ aliphatic wherein two methylene units are optionally andindependently replaced by -Cy- or —C(O)—. In some embodiments, R^(L2) isan optionally substituted C₁-C₉ aliphatic wherein two methylene unitsare optionally and independently replaced by -Cy- or —C(O)—. ExampleR^(L2) groups are depicted below:

In some embodiments, R¹ is hydrogen, or an optionally substituted groupselected from

—S—(C₁-C₁₀ aliphatic), C₁-C₁₀ aliphatic, aryl, C₁-C₆ heteroalkyl,heteroaryl and heterocyclyl. In some embodiments, R¹ is

or —S—(C₁-C₁₀ aliphatic). In some embodiments, R¹ is

In some embodiments, R¹ is an optionally substituted group selected from—S—(C₁-C₆ aliphatic), C₁-C₁₀ aliphatic, C₁-C₆ heteroaliphatic, aryl,heterocyclyl and heteroaryl.

In some embodiments, R¹ is

In some embodiments, the sulfur atom in the R¹ embodiments describedabove and herein is connected with the sulfur atom, G, E, or —C(O)—moiety in the L embodiments described above and herein. In someembodiments, the —C(O)— moiety in the R¹ embodiments described above andherein is connected with the sulfur atom, G, E, or —C(O)— moiety in theL embodiments described above and herein.

In some embodiments, -L-R¹ is any combination of the L embodiments andR¹ embodiments described above and herein.

In some embodiments, -L-R¹ is -L³-G-R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L⁴-G-R¹ wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L³-G-S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ is -L³-G-C(O)—R^(L2), wherein each variableis independently as defined above and described herein.

In some embodiments, -L-R¹ is

wherein R^(L2) is an optionally substituted C₁-C₉ aliphatic wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—,or —C(O)O—, and each G is independently as defined above and describedherein.

In some embodiments, -L-R¹ is —R^(L3)—S—S—R^(L2), wherein each variableis independently as defined above and described herein. In someembodiments, -L-R¹ is —R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, -L-R¹ has the structure of:

wherein each variable is independently as defined above and describedherein.

In some embodiments, —X-L-R¹ has the structure of:

wherein:the phenyl ring is optionally substituted, andeach of R¹ and X is independently as defined above and described herein.

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ is:

In some embodiments, -L-R¹ is CH₃—,

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ comprises a terminal optionally substituted—(CH₂)₂— moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)₂— moiety which is connected to X. Examplesuch -L-R¹ moieties are depicted below:

In some embodiments, -L-R¹ comprises a terminal optionally substituted—(CH₂)— moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)— moiety which is connected to X. Examplesuch -L-R¹ moieties are depicted below:

In some embodiments, -L-R¹ is

In some embodiments, -L-R¹ is CH₃—,

and X is —S—.

In some embodiments, -L-R¹ is CH₃—,

X is —S—, W is O, Y is —O—, and Z is —O—.

In some embodiments, R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, R¹ is

In some embodiments, X is —O— or —S—, and R¹ is

or —S—(C₁-C₁₀ aliphatic).

In some embodiments, X is —O— or —S—, and R¹ is

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, L is a covalent bond and -L-R¹ is R¹.

In some embodiments, -L-R¹ is not hydrogen.

In some embodiments, —X-L-R¹ is R¹ is

—S—(C₁-C₁₀ aliphatic) or —S—(C₁-C₅₀ aliphatic).

In some embodiments, —X-L-R¹ has the structure of

wherein the

moiety is optionally substituted. In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ is

In some embodiments, —X-L-R¹ has the structure of

wherein X′ is O or S, Y′ is —O—, —S— or —NR′—, and the

moiety is optionally substituted. In some embodiments, Y′ is —O—, —S— or—NH—. In some embodiments,

In some embodiments,

In some embodiments,

In some embodiments, —X-L-R¹ has the structure of

wherein X′ is O or S, and the

moiety is optionally substituted. In some embodiments,

In some embodiments, —X-L-R¹ is

wherein the

is optionally substituted. In some embodiments, —X-L-R¹ is

wherein the

is substituted. In some embodiments, —X-L-R¹ is

wherein the

is unsubstituted.

In some embodiments, —X-L-R¹ is R¹—C(O)—S-L^(X)-S—, wherein L^(x) is anoptionally substituted group selected from

In some embodiments, L^(x) is

In some embodiments, —X-L-R¹ is (CH₃)₃C—S—S-L^(x)-S—. In someembodiments, —X-L-R¹ is R¹—C(═X′)—Y′—C(R)₂—S-L^(x)-S—. In someembodiments, —X-L-R¹ is R—C(═X′)—Y′—CH₂—S-L^(x)-S⁻. In some embodiments,—X-L-R¹ is

As will be appreciated by a person skilled in the art, many of the—X-L-R¹ groups described herein are cleavable and can be converted to—X— after administration to a subject. In some embodiments, —X-L-R¹ iscleavable. In some embodiments, —X-L-R¹ is —S-L-R¹, and is converted to—S— after administration to a subject. In some embodiments, theconversion is promoted by an enzyme of a subject. As appreciated by aperson skilled in the art, methods of determining whether the —S-L-R¹group is converted to —S— after administration is widely known andpracticed in the art, including those used for studying drug metabolismand pharmacokinetics.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-a:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I has thestructure of formula I-b:

wherein each variable is independently as defined above and describedherein.

In some embodiments, the internucleotidic linkage of formula I is anphosphorothioate triester linkage having the structure of formula I-c:

wherein:

-   P* is an asymmetric phosphorus atom and is either Rp or Sp;-   L is a covalent bond or an optionally substituted, linear or    branched C₁-C₁₀ alkylene, wherein one or more methylene units of L    are optionally and independently replaced by an optionally    substituted C₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, —C(R′)₂—, -Cy-,    —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—,    —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or    —C(O)O—;-   R¹ is halogen, R, or an optionally substituted C₁-C₅₀ aliphatic    wherein one or more methylene units are optionally and independently    replaced by an optionally substituted C₁-C₆ alkylene, C₁-C₆    alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—,    —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—,    —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—,    —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;-   each R′ is independently —R, —C(O)R, —C₀₂R, or —SO₂R, or:    -   two R′ on the same nitrogen are taken together with their        intervening atoms to form an optionally substituted heterocyclic        or heteroaryl ring, or    -   two R′ on the same carbon are taken together with their        intervening atoms to form an optionally substituted aryl,        carbocyclic, heterocyclic, or heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    phenylene, carbocyclylene, arylene, heteroarylene, or    heterocyclylene;-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl;-   each

independently represents a connection to a nucleoside; and

-   R¹ is not —H when L is a covalent bond.

In some embodiments, the internucleotidic linkage having the structureof formula I is

In some embodiments, the internucleotidic linkage having the structureof formula I-c is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising one or more phosphate diesterlinkages, and one or more modified internucleotide linkages having theformula of I-a, I-b, or I-c.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least one phosphorothioate triesterlinkage having the structure of formula I-c. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least two phosphorothioate triester linkages having the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising at least one phosphatediester internucleotidic linkage and at least three phosphorothioatetriester linkages having the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising at least one phosphate diesterinternucleotidic linkage and at least four phosphorothioate triesterlinkages having the structure of formula I-c. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising at least one phosphate diester internucleotidic linkage andat least five phosphorothioate triester linkages having the structure offormula I-c.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 38). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising asequence found in GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the saidsequence has over 50% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO:38). In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the said sequence has over60% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO: 38). In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 38), wherein the said sequence has over 70% identity withGGCACAAGGGCACAGACTTC (SEQ ID NO: 38). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising asequence found in GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the saidsequence has over 80% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO:38). In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the said sequence has over90% identity with GGCACAAGGGCACAGACTTC (SEQ ID NO: 38). In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 38), wherein the said sequence has over 95% identity withGGCACAAGGGCACAGACTTC (SEQ ID NO: 38). In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38). In some embodiments,the present disclosure provides a chirally controlled oligonucleotidehaving the sequence of GGCACAAGGGCACAGACTTC.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 38), wherein at least one internucleotidic linkage has thestructure of formula I. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide comprising a sequencefound in GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein eachinternucleotidic linkage has the structure of formula I. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 38), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide comprising a sequencefound in GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein eachinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising a sequence found in GGCACAAGGGCACAGACTTC (SEQID NO: 38), wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein each internucleotidiclinkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least oneinternucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence found inGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein each internucleotidiclinkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least oneinternucleotidic linkage has a chiral linkage phosphorus. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising the sequence of GGCACAAGGGCACAGACTTC (SEQ IDNO: 38), wherein at least one internucleotidic linkage has the structureof formula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein each internucleotidiclinkage has the structure of formula I. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide comprising thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least oneinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide comprising the sequence of GGCACAAGGGCACAGACTTC (SEQ IDNO: 38), wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least oneinternucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein each internucleotidiclinkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least oneinternucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein each internucleotidiclinkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein at least one internucleotidic linkage has achiral linkage phosphorus. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least oneinternucleotidic linkage has the structure of formula I. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:38), wherein each internucleotidic linkage has the structure of formulaI. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein at least one internucleotidic linkage has thestructure of formula I-c. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein each internucleotidiclinkage has the structure of formula I-c. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at leastone internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein at least one linkage phosphorus is Rp. It isunderstood by a person of ordinary skill in the art that in certainembodiments wherein the chirally controlled oligonucleotide comprises anRNA sequence, each T is independently and optionally replaced with U. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:38), wherein each linkage phosphorus is Rp. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at leastone linkage phosphorus is Sp. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein each linkagephosphorus is Sp. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the oligonucleotide is ablockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the oligonucleotide is astereoblockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the oligonucleotide is aP-modification blockmer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the oligonucleotide is alinkage blockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the oligonucleotide is analtmer. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein the oligonucleotide is a stereoaltmer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:38), wherein the oligonucleotide is a P-modification altmer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:38), wherein the oligonucleotide is a linkage altmer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having the sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO:38), wherein the oligonucleotide is a unimer. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein theoligonucleotide is a stereounimer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein theoligonucleotide is a P-modification unimer. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingthe sequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein theoligonucleotide is a linkage unimer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein theoligonucleotide is a gapmer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having the sequence ofGGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein the oligonucleotide is askipmer.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein each cytosine is optionally and independentlyreplaced by 5-methylcytosine. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having thesequence of GGCACAAGGGCACAGACTTC (SEQ ID NO: 38), wherein at least onecytosine is optionally and independently replaced by 5-methylcytosine.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having the sequence of GGCACAAGGGCACAGACTTC(SEQ ID NO: 38), wherein each cytosine is optionally and independentlyreplaced by 5-methylcytosine.

In some embodiments, a chirally controlled oligonucleotide is designedsuch that one or more nucleotides comprise a phosphorus modificationprone to “autorelease” under certain conditions. That is, under certainconditions, a particular phosphorus modification is designed such thatit self-cleaves from the oligonucleotide to provide, e.g., a phosphatediester such as those found in naturally occurring DNA and RNA. In someembodiments, such a phosphorus modification has a structure of —O-L-R¹,wherein each of L and R¹ is independently as defined above and describedherein. In some embodiments, an autorelease group comprises a morpholinogroup. In some embodiments, an autorelease group is characterized by theability to deliver an agent to the internucleotidic phosphorus linker,which agent facilitates further modification of the phosphorus atom suchas, e.g., desulfurization. In some embodiments, the agent is water andthe further modification is hydrolysis to form a phosphate diester as isfound in naturally occurring DNA and RNA.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein(including, as non-limiting examples, any sequence disclosed in anyTable). In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising a sequence having over 50%identity with any sequence disclosed herein. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotidecomprising a sequence having over 60% identity with any sequencedisclosed herein. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising a sequence having over70% identity with any sequence disclosed herein. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomprising a sequence having over 80% identity with any sequencedisclosed herein. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising a sequence having over90% identity with any sequence disclosed herein. In some embodiments,the present disclosure provides a chirally controlled oligonucleotidecomprising a sequence having over 95% identity with any sequencedisclosed herein. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage has a chiral linkagephosphorus. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage has a chiral linkagephosphorus. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage has the structureof formula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein each internucleotidic linkage has the structure offormula I-c. In some embodiments, the present disclosure provides achirally controlled oligonucleotide comprising any sequence disclosedherein, wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein at least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide comprising any sequence disclosed herein,wherein each internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one internucleotidic linkage has a chiral linkage phosphorus.In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one internucleotidic linkage has the structure of formula I. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein eachinternucleotidic linkage has the structure of formula I. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein at leastone internucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein eachinternucleotidic linkage has the structure of formula I-c. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein at leastone internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereineach internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereineach internucleotidic linkage is

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinat least one linkage phosphorus is Rp. It is understood by a person ofordinary skill in the art that in certain embodiments wherein thechirally controlled oligonucleotide comprises an RNA sequence, each T isindependently and optionally replaced with U. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingany sequence disclosed herein, wherein each linkage phosphorus is Rp. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein at leastone linkage phosphorus is Sp. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein each linkage phosphorus is Sp. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a blockmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is astereoblockmer. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having any sequence disclosedherein, wherein the oligonucleotide is a P-modification blockmer. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a linkage blockmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is an altmer. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a stereoaltmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is aP-modification altmer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having any sequencedisclosed herein, wherein the oligonucleotide is a linkage altmer. Insome embodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a unimer. In some embodiments, the present disclosureprovides a chirally controlled oligonucleotide having any sequencedisclosed herein, wherein the oligonucleotide is a stereounimer. In someembodiments, the present disclosure provides a chirally controlledoligonucleotide having any sequence disclosed herein, wherein theoligonucleotide is a P-modification unimer. In some embodiments, thepresent disclosure provides a chirally controlled oligonucleotide havingany sequence disclosed herein, wherein the oligonucleotide is a linkageunimer. In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereinthe oligonucleotide is a gapmer. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein the oligonucleotide is a skipmer.

In some embodiments, the present disclosure provides a chirallycontrolled oligonucleotide having any sequence disclosed herein, whereineach cytosine is optionally and independently replaced by5-methylcytosine. In some embodiments, the present disclosure provides achirally controlled oligonucleotide having any sequence disclosedherein, wherein at least one cytosine is optionally and independentlyreplaced by 5-methylcytosine. In some embodiments, the presentdisclosure provides a chirally controlled oligonucleotide having anysequence disclosed herein, wherein each cytosine is optionally andindependently replaced by 5-methylcytosine.

In various embodiments, any sequence disclosed herein can be combinedwith one or more of the following as disclosed herein or known in theart: pattern of backbone linkages; pattern of backbone chiral centers;and pattern of backbone P-modifications; pattern of base modification;pattern of sugar modification; pattern of backbone linkages; pattern ofbackbone chiral centers; and pattern of backbone P-modifications.

In some embodiments, a chirally controlled oligonucleotide is designedsuch that the resulting pharmaceutical properties are improved throughone or more particular modifications at phosphorus. It is welldocumented in the art that certain oligonucleotides are rapidly degradedby nucleases and exhibit poor cellular uptake through the cytoplasmiccell membrane (Poijarvi-Virta et al., Curr. Med. Chem. (2006), 13(28);3441-65; Wagner et al., Med. Res. Rev. (2000), 20(6):417-51; Peyrotteset al., Mini Rev. Med. Chem. (2004), 4(4):395-408; Gosselin et al.,(1996), 43(1):196-208; Bologna et al., (2002), Antisense & Nucleic AcidDrug Development 12:33-41). For instance, Vives et al., (Nucleic AcidsResearch (1999), 27(20):4071-76) found that tert-butyl SATEpro-oligonucleotides displayed markedly increased cellular penetrationcompared to the parent oligonucleotide.

In some embodiments, a modification at a linkage phosphorus ischaracterized by its ability to be transformed to a phosphate diester,such as those present in naturally occurring DNA and RNA, by one or moreesterases, nucleases, and/or cytochrome P450 enzymes, including but notlimited to, those listed in Table 1A, below.

TABLE 1A Example enzymes. Family Gene CYP1 CYP1A1, CYP1A2, CYP1B1 CYP2CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19,CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 CYP3CYP3A4, CYP3A5, CYP3A7, CYP3A43 CYP4 CYP4A11, CYP4A22, CYP4B1, CYP4F2,CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1 CYP5CYP5A1 CYP7 CYP7A1, CYP7B1 CYP8 CYP8A1 (prostacyclin synthase), CYP8B1(bile acid biosynthesis) CYP11 CYP11A1, CYP11B1, CYP11B2 CYP17 CYP17A1CYP19 CYP19A1 CYP20 CYP20A1 CYP21 CYP21A2 CYP24 CYP24A1 CYP26 CYP26A1,CYP26B1, CYP26C1 CYP27 CYP27A1 (bile acid biosynthesis), CYP27B1(vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknownfunction) CYP39 CYP39A1 CYP46 CYP46A1 CYP51 CYP51A1 (lanosterol 14-alphademethylase)

In some embodiments, a modification at phosphorus results in aP-modification moiety characterized in that it acts as a pro-drug, e.g.,the P-modification moiety facilitates delivery of an oligonucleotide toa desired location prior to removal. For instance, in some embodiments,a P-modification moiety results from PEGylation at the linkagephosphorus. One of skill in the relevant arts will appreciate thatvarious PEG chain lengths are useful and that the selection of chainlength will be determined in part by the result that is sought to beachieved by PEGylation. For instance, in some embodiments, PEGylation iseffected in order to reduce RES uptake and extend in vivo circulationlifetime of an oligonucleotide.

In some embodiments, a PEGylation reagent for use in accordance with thepresent disclosure is of a molecular weight of about 300 g/mol to about100,000 g/mol. In some embodiments, a PEGylation reagent is of amolecular weight of about 300 g/mol to about 10,000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 300g/mol to about 5,000 g/mol. In some embodiments, a PEGylation reagent isof a molecular weight of about 500 g/mol. In some embodiments, aPEGylation reagent of a molecular weight of about 1000 g/mol. In someembodiments, a PEGylation reagent is of a molecular weight of about 3000g/mol. In some embodiments, a PEGylation reagent is of a molecularweight of about 5000 g/mol.

In certain embodiments, a PEGylation reagent is PEG500. In certainembodiments, a PEGylation reagent is PEG1000. In certain embodiments, aPEGylation reagent is PEG3000. In certain embodiments, a PEGylationreagent is PEG5000.

In some embodiments, a P-modification moiety is characterized in that itacts as a PK enhancer, e.g., lipids, PEGylated lipids, etc.

In some embodiments, a P-modification moiety is characterized in that itacts as an agent which promotes cell entry and/or endosomal escape, suchas a membrane-disruptive lipid or peptide.

In some embodiments, a P-modification moiety is characterized in that itacts as a targeting agent. In some embodiments, a P-modification moietyis or comprises a targeting agent. The phrase “targeting agent,” as usedherein, is an entity that is associates with a payload of interest(e.g., with an oligonucleotide or oligonucleotide composition) and alsointeracts with a target site of interest so that the payload of interestis targeted to the target site of interest when associated with thetargeting agent to a materially greater extent than is observed underotherwise comparable conditions when the payload of interest is notassociated with the targeting agent. A targeting agent may be, orcomprise, any of a variety of chemical moieties, including, for example,small molecule moieties, nucleic acids, polypeptides, carbohydrates,etc. Targeting agents are described further by Adarsh et al., “OrganelleSpecific Targeted Drug Delivery—A Review,” International Journal ofResearch in Pharmaceutical and Biomedical Sciences, 2011, p. 895.

Example such targeting agents include, but are not limited to, proteins(e.g. Transferrin), oligopeptides (e.g., cyclic and acylicRGD-containing oligopedptides), antibodies (monoclonal and polyclonalantibodies, e.g. IgG, IgA, IgM, IgD, IgE antibodies),sugars/carbohydrates (e.g., monosaccharides and/or oligosaccharides(mannose, mannose-6-phosphate, galactose, and the like)), vitamins(e.g., folate), or other small biomolecules. In some embodiments, atargeting moiety is a steroid molecule (e.g., bile acids includingcholic acid, deoxycholic acid, dehydrocholic acid; cortisone;digoxigenin; testosterone; cholesterol; cationic steroids such ascortisone having a trimethylaminomethyl hydrazide group attached via adouble bond at the 3-position of the cortisone ring, etc.). In someembodiments, a targeting moiety is a lipophilic molecule (e.g.,alicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes,terpenes, and polyalicyclic hydrocarbons such as adamantine andbuckminsterfullerenes). In some embodiments, a lipophilic molecule is aterpenoid such as vitamin A, retinoic acid, retinal, or dehydroretinal.In some embodiments, a targeting moiety is a peptide.

In some embodiments, a P-modification moiety is a targeting agent offormula —X-L-R¹ wherein each of X, L, and R¹ are as defined in Formula Iabove.

In some embodiments, a P-modification moiety is characterized in that itfacilitates cell specific delivery.

In some embodiments, a P-modification moiety is characterized in that itfalls into one or more of the above-described categories. For instance,in some embodiments, a P-modification moiety acts as a PK enhancer and atargeting ligand. In some embodiments, a P-modification moiety acts as apro-drug and an endosomal escape agent. One of skill in the relevantarts would recognize that numerous other such combinations are possibleand are contemplated by the present disclosure.

Nucleobases

In some embodiments, a nucleobase present in a provided oligonucleotideis a natural nucleobase or a modified nucleobase derived from a naturalnucleobase. Examples include, but are not limited to, uracil, thymine,adenine, cytosine, and guanine having their respective amino groupsprotected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine,5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidineanalogs such as pseudoisocytosine and pseudouracil and other modifiednucleobases such as 8-substituted purines, xanthine, or hypoxanthine(the latter two being the natural degradation products). Examplemodified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9,1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7,313. In some embodiments, a modified nucleobase is substituted uracil,thymine, adenine, cytosine, or guanine. In some embodiments, a modifiednucleobase is a functional replacement, e.g., in terms of hydrogenbonding and/or base pairing, of uracil, thymine, adenine, cytosine, orguanine. In some embodiments, a nucleobase is optionally substituteduracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine. Insome embodiments, a nucleobase is uracil, thymine, adenine, cytosine,5-methylcytosine, or guanine.

Compounds represented by the following general formulae are alsocontemplated as modified nucleobases:

wherein R⁸ is an optionally substituted, linear or branched groupselected from aliphatic, aryl, aralkyl, aryloxylalkyl, carbocyclyl,heterocyclyl or heteroaryl group having 1 to 15 carbon atoms, including,by way of example only, a methyl, isopropyl, phenyl, benzyl, orphenoxymethyl group; and each of R⁹ and R¹⁰ is independently anoptionally substituted group selected from linear or branched aliphatic,carbocyclyl, aryl, heterocyclyl and heteroaryl.

Modified nucleobases also include expanded-size nucleobases in which oneor more aryl rings, such as phenyl rings, have been added. Nucleic basereplacements described in the Glen Research catalog(www.glenresearch.com); Krueger A T et al, Acc. Chem. Res., 2007, 40,141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., etal., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr.Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol.,2006, 10, 622-627, are contemplated as useful for the synthesis of thenucleic acids described herein. Some examples of these expanded-sizenucleobases are shown below:

Herein, modified nucleobases also encompass structures that are notconsidered nucleobases but are other moieties such as, but not limitedto, corrin- or porphyrin-derived rings. Porphyrin-derived basereplacements have been described in Morales-Rojas, H and Kool, E T, Org.Lett., 2002, 4, 4377-4380. Shown below is an example of aporphyrin-derived ring which can be used as a base replacement:

In some embodiments, modified nucleobases are of any one of thefollowing structures, optionally substituted:

In some embodiments, a modified nucleobase is fluorescent. Example suchfluorescent modified nucleobases include phenanthrene, pyrene,stillbene, isoxanthine, isozanthopterin, terphenyl, terthiophene,benzoterthiophene, coumarin, lumazine, tethered stillbene, benzo-uracil,and naphtho-uracil, as shown below:

In some embodiments, a modified nucleobase is unsubstituted. In someembodiments, a modified nucleobase is substituted. In some embodiments,a modified nucleobase is substituted such that it contains, e.g.,heteroatoms, alkyl groups, or linking moieties connected to fluorescentmoieties, biotin or avidin moieties, or other protein or peptides. Insome embodiments, a modified nucleobase is a “universal base” that isnot a nucleobase in the most classical sense, but that functionssimilarly to a nucleobase. One representative example of such auniversal base is 3-nitropyrrole.

In some embodiments, other nucleosides can also be used in the processdisclosed herein and include nucleosides that incorporate modifiednucleobases, or nucleobases covalently bound to modified sugars. Someexamples of nucleosides that incorporate modified nucleobases include4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2′-O-methylcytidine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; dihydrouridine;2′-O-methylpseudouridine; beta,D-galactosylqueosine;2′-O-methylguanosine; N⁶-isopentenyladenosine; 1-methyladenosine;1-methylpseudouridine; 1-methylguanosine; 1-methylinosine;2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine;N⁷-methylguanosine; 3-methyl-cytidine; 5-methylcytidine;5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine;N⁶-methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine;5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine;5-methoxycarbonylmethyluridine; 5-methoxyuridine;2-methylthio-N⁶-isopentenyladenosine;N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine;N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine;uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v);pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine;2-thiouridine; 4-thiouridine; 5-methyluridine;2′-O-methyl-5-methyluridine; and 2′-O-methyluridine.

In some embodiments, nucleosides include 6′-modified bicyclic nucleosideanalogs that have either (R) or (S)-chirality at the 6′-position andinclude the analogs described in U.S. Pat. No. 7,399,845. In otherembodiments, nucleosides include 5′-modified bicyclic nucleoside analogsthat have either (R) or (S)-chirality at the 5′-position and include theanalogs described in US Patent Application Publication No. 20070287831.

In some embodiments, a nucleobase or modified nucleobase comprises oneor more biomolecule binding moieties such as e.g., antibodies, antibodyfragments, biotin, avidin, streptavidin, receptor ligands, or chelatingmoieties. In other embodiments, a nucleobase or modified nucleobase is5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments,a nucleobase or modified nucleobase is modified by substitution with afluorescent or biomolecule binding moiety. In some embodiments, thesubstituent on a nucleobase or modified nucleobase is a fluorescentmoiety. In some embodiments, the substituent on a nucleobase or modifiednucleobase is biotin or avidin.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200;6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;6,617,438; 7,045,610; 7,427,672; and 7,495,088, the modifiednucleobases, sugars, and internucleotidic linkages of each of which areincorporated by reference.

In some embodiments, a base is optionally substituted A, T, C, G or U,wherein one or more —NH₂ are independently and optionally replaced with—C(-L-R¹)₃, one or more —NH— are independently and optionally replacedwith —C(-L-R¹)₂—, one or more ═N— are independently and optionallyreplaced with —C(-L-R¹)—, one or more ═CH— are independently andoptionally replaced with ═N—, and one or more ═O are independently andoptionally replaced with ═S, ═N(-L-R¹), or ═C(-L-R¹)₂, wherein two ormore -L-R¹ are optionally taken together with their intervening atoms toform a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatomring atoms. In some embodiments, a modified base is optionallysubstituted A, T, C, G or U, wherein one or more —NH₂ are independentlyand optionally replaced with —C(-L-R¹)₃, one or more —NH— areindependently and optionally replaced with —C(-L-R¹)₂—, one or more ═N—are independently and optionally replaced with —C(-L-R¹)—, one or more═CH— are independently and optionally replaced with ═N—, and one or more═O are independently and optionally replaced with ═S, ═N(-L-R¹), or═C(-L-R¹)₂, wherein two or more -L-R¹ are optionally taken together withtheir intervening atoms to form a 3-30 membered bicyclic or polycyclicring having 0-10 heteroatom ring atoms, wherein the modified base isdifferent than the natural A, T, C, G and U. In some embodiments, a baseis optionally substituted A, T, C, G or U. In some embodiments, amodified base is substituted A, T, C, G or U, wherein the modified baseis different than the natural A, T, C, G and U.

In some embodiments, a modified nucleotide or nucleotide analog is anymodified nucleotide or nucleotide analog described in any of: Gryaznov,S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsenet al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58,2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Koshkin et al.1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo. Med. Chem. Let.8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen etal. 2003 Bioo. Med. Chem. Lett. 13: 253-256; Mesmaeker et al. Angew.Chem., Int. Ed. Engl. 1994, 33, 226; Morita et al. 2001 Nucl. Acids Res.Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76;Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al. 1997Chem. Soc. Rev. 73; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1:3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika etal. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan et al. 2012 Chem. Comm.48: 8195-8197; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshiet al. 1999 Chem. Commun. 1395-1396; Schultz et al. 1996 Nucleic AcidsRes. 24: 2966; Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al.2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2010 J. Org. Chem. 75:1569-1581; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth etal. 2012 Mol. Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew;Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Thazha P;Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; etal. From Nucleic Acids Symposium Series (2008), 52(1), 553-554; Singh etal. 1998 Chem. Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63:10035-39; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003Chem. Comm. 2130-2131; Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220;Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseuret al. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO20070900071; or WO 2016/079181.

Sugars

In some embodiments, provided oligonucleotides comprise one or moremodified sugar moieties beside the natural sugar moieties.

The most common naturally occurring nucleotides are comprised of ribosesugars linked to the nucleobases adenosine (A), cytosine (C), guanine(G), and thymine (T) or uracil (U). Also contemplated are modifiednucleotides wherein a phosphate group or linkage phosphorus in thenucleotides can be linked to various positions of a sugar or modifiedsugar. As non-limiting examples, the phosphate group or linkagephosphorus can be linked to the 2′, 3′, 4′ or 5′ hydroxyl moiety of asugar or modified sugar. Nucleotides that incorporate modifiednucleobases as described herein are also contemplated in this context.In some embodiments, nucleotides or modified nucleotides comprising anunprotected —OH moiety are used in accordance with methods of thepresent disclosure.

Other modified sugars can also be incorporated within a providedoligonucleotide. In some embodiments, a modified sugar contains one ormore substituents at the 2′ position including one of the following: —F;—CF₃, —CN, —N3, —NO, —NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ isindependently as defined above and described herein; —O—(C₁-C₁₀ alkyl),—S—(C₁-C₁₀ alkyl), —NH—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀alkenyl), —S—(C₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀alkenyl)₂; —O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀alkynyl), or —N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀alkyl), —O—(C₁-C₁₀ alkylene)-NH—(C₁-C₁₀ alkyl) or —O—(C₁-C₁₀alkylene)-NH(C₁-C₁₀ alkyl)₂, —NH—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), or—N(C₁-C₁₀ alkyl)-(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), wherein the alkyl,alkylene, alkenyl and alkynyl may be substituted or unsubstituted.Examples of substituents include, and are not limited to,—O(CH₂)_(n)OCH₃, and —O(CH₂)_(n)NH₂, wherein n is from 1 to about 10,MOE, DMAOE, DMAEOE. Also contemplated herein are modified sugarsdescribed in WO 2001/088198; and Martin et al., Helv. Chim. Acta, 1995,78, 486-504. In some embodiments, a modified sugar comprises one or moregroups selected from a substituted silyl group, an RNA cleaving group, areporter group, a fluorescent label, an intercalator, a group forimproving the pharmacokinetic properties of a nucleic acid, a group forimproving the pharmacodynamic properties of a nucleic acid, or othersubstituents having similar properties. In some embodiments,modifications are made at one or more of the the 2′, 3′, 4′, 5′, or 6′positions of the sugar or modified sugar, including the 3′ position ofthe sugar on the 3′-terminal nucleotide or in the 5′ position of the5′-terminal nucleotide.

In some embodiments, the 2′-OH of a ribose is replaced with asubstituent including one of the following: —H, —F; —CF₃, —CN, —N3, —NO,—NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ is independently asdefined above and described herein; —O—(C₁-C₁₀ alkyl), —S—(C₁-C₁₀alkyl), —NH—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀ alkyl)₂; —O—(C₂-C₁₀ alkenyl),—S—(C₂-C₁₀ alkenyl), —NH—(C₂-C₁₀ alkenyl), or —N(C₂-C₁₀ alkenyl)₂;—O—(C₂-C₁₀ alkynyl), —S—(C₂-C₁₀ alkynyl), —NH—(C₂-C₁₀ alkynyl), or—N(C₂-C₁₀ alkynyl)₂; or —O—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl),—O—(C₁-C₁₀ alkylene)-NH—(C₁-C₁₀ alkyl) or —O—(C₁-C₁₀ alkylene)-NH(C₁-C₁₀alkyl)₂, —NH—(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), or —N(C₁-C₁₀alkyl)-(C₁-C₁₀ alkylene)-O—(C₁-C₁₀ alkyl), wherein the alkyl, alkylene,alkenyl and alkynyl may be substituted or unsubstituted. In someembodiments, the 2′-OH is replaced with —H (deoxyribose). In someembodiments, the 2′-OH is replaced with —F. In some embodiments, the2′-OH is replaced with —OR′. In some embodiments, the 2′-OH is replacedwith —OMe. In some embodiments, the 2′-OH is replaced with —OCH₂CH₂OMe.

Modified sugars also include locked nucleic acids (LNAs). In someembodiments, two substituents on sugar carbon atoms are taken togetherto form a bivalent moiety. In some embodiments, two substituents are ontwo different sugar carbon atoms. In some embodiments, a formed bivalentmoiety has the structure of -L- as defined herein. In some embodiments,-L- is —O—CH₂—, wherein —CH₂— is optionally substituted. In someembodiments, -L- is —O—CH₂—. In some embodiments, -L- is —O—CH(Et)-. Insome embodiments, -L- is between C₂ and C₄ of a sugar moiety. In someembodiments, a locked nucleic acid has the structure indicated below. Alocked nucleic acid of the structure below is indicated, wherein Barepresents a nucleobase or modified nucleobase as described herein, andwherein R^(2s) is —OCH₂C4′-.

In some embodiments, a modified sugar is an ENA such as those describedin, e.g., Seth et al., J Am Chem Soc. 2010 Oct. 27; 132(42):14942-14950. In some embodiments, a modified sugar is any of those foundin an XNA (xenonucleic acid), for instance, arabinose, anhydrohexitol,threose, 2′fluoroarabinose, or cyclohexene.

Modified sugars include sugar mimetics such as cyclobutyl or cyclopentylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; and 5,359,044. Some modified sugars that arecontemplated include sugars in which the oxygen atom within the ribosering is replaced by nitrogen, sulfur, selenium, or carbon. In someembodiments, a modified sugar is a modified ribose wherein the oxygenatom within the ribose ring is replaced with nitrogen, and wherein thenitrogen is optionally substituted with an alkyl group (e.g., methyl,ethyl, isopropyl, etc).

Non-limiting examples of modified sugars include glycerol, which formglycerol nucleic acid (GNA) analogues. One example of a GNA analogue isshown below and is described in Zhang, R et al., J. Am. Chem. Soc.,2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc., 2005, 127,4174-4175 and Tsai C H et al., PNAS, 2007, 14598-14603 (X═O—):

Another example of a GNA derived analogue, flexible nucleic acid (FNA)based on the mixed acetal aminal of formyl glycerol, is described inJoyce G F et al., PNAS, 1987, 84, 4398-4402 and Heuberger B D andSwitzer C, J. Am. Chem. Soc., 2008, 130, 412-413, and is shown below:

Additional non-limiting examples of modified sugars includehexopyranosyl (6′ to 4′), pentopyranosyl (4′ to 2′), pentopyranosyl (4′to 3′), or tetrofuranosyl (3′ to 2′) sugars. In some embodiments, ahexopyranosyl (6′ to 4′) sugar is of any one in the following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 2′) sugar is of any one inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a pentopyranosyl (4′ to 3′) sugar is of any one inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a tetrofuranosyl (3′ to 2′) sugar is of either inthe following formulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, a modified sugar is of any one in the followingformulae:

wherein X^(s) corresponds to the P-modification group “—XLR¹” describedherein and Ba is as defined herein.

In some embodiments, one or more hydroxyl group in a sugar moiety isoptionally and independently replaced with halogen, R′ —N(R′)₂, —OR′, or—SR′, wherein each R′ is independently as defined above and describedherein.

In some embodiments, a sugar mimetic is as illustrated below, whereinX^(s) corresponds to the P-modification group “—XLR¹” described herein,Ba is as defined herein, and X¹ is selected from —S—, —Se—, —CH₂—,—NMe-, -NEt- or —NiPr—.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more(e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%0 or more), inclusive,of the sugars in a chirally controlled oligonucleotide composition aremodified. In some embodiments, only purine residues are modified (e.g.,about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more] of the purine residues are modified).In some embodiments, only pyrimidine residues are modified (e.g., about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more] of the pyridimine residues are modified). Insome embodiments, both purine and pyrimidine residues are modified.

Modified sugars and sugar mimetics can be prepared by methods known inthe art, including, but not limited to: A. Eschenmoser, Science (1999),284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75:1416-1477; M.Egli et al, J. Am. Chem. Soc. (2006), 128(33):10847-56; A. Eschenmoserin Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V.Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p. 293; K.-U.Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et al,Helv. Chim. Acta (1992), 75:218; J. Hunziker et al, Helv. Chim. Acta(1993), 76:259; G. Otting et al, Helv. Chim. Acta (1993), 76:2701; K.Groebke et al, Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser,Science (1999), 284:2118. Modifications to the 2′ modifications can befound in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and allreferences therein. Specific modifications to the ribose can be found inthe following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem.,1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79,1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310). Insome embodiments, a modified sugar is any of those described in PCTPublication No. WO2012/030683, incorporated herein by reference, anddepicted in the FIGS. 26-30 of the present application.

In some embodiments, a modified sugar moiety is an optionallysubstituted pentose or hexose moiety. In some embodiments, a modifiedsugar moiety is an optionally substituted pentose moiety. In someembodiments, a modified sugar moiety is an optionally substituted hexosemoiety. In some embodiments, a modified sugar moiety is an optionallysubstituted ribose or hexitol moiety. In some embodiments, a modifiedsugar moiety is an optionally substituted ribose moiety. In someembodiments, a modified sugar moiety is an optionally substitutedhexitol moiety.

In some embodiments, an example modified internucleotidic linkage and/orsugar is selected from:

In some embodiments, R¹ is R as defined and described. In someembodiments, R² is R. In some embodiments, R^(e) is R. In someembodiments, R^(e) is H, CH₃, Bn, COCF₃, benzoyl, benzyl,pyren-1-ylcarbonyl, pyren-1-ylmethyl, 2-aminoethyl. In some embodiments,an example modified internucleotidic linkage and/or sugar is selectedfrom those described in Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507,220; Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143;Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Jones etal. J. Org. Chem. 1993, 58, 2983; Vasseur et al. J. Am. Chem. Soc. 1992,114, 4006; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34:1338; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Koshkin et al. 1998Tetrahedron 54: 3607-3630; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5;Nielsen et al. 1997 Chem. Soc. Rev. 73; Schultz et al. 1996 NucleicAcids Res. 24: 2966; Obika et al. 1997 Tetrahedron Lett. 38 (50):8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Singh et al.1998 Chem. Comm. 1247-1248; Kumar et al. 1998 Bioo. Med. Chem. Let. 8:2219-2222; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1:3423-3433; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Seth et al.2010 J. Org. Chem. 75: 1569-1581; Singh et al. 1998 J. Org. Chem. 63:10035-39; Sorensen 2003 Chem. Comm. 2130-2131; Petersen et al. 2003TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun.1395-1396; Jepsen et al. 2004 Oligo. 14: 130-146; Morita et al. 2001Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem.Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226;Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Lauritsen et al. 2002Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13:253-256; WO 20070900071; Seth et al., Nucleic Acids Symposium Series(2008), 52(1), 553-554; Seth et al. 2009 J. Med. Chem. 52: 10-13; Sethet al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Pallan et al. 2012 Chem. Comm.48: 8195-8197; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al.2012 Bioo. Med. Chem. Lett. 22: 296-299; WO 2016/079181; U.S. Pat. Nos.6,326,199; 6,066,500; and 6,440,739, the base and sugar modifications ofeach of which is herein incorporated by reference.

Oligonucleotides

In some embodiments, the present disclosure provides oligonucleotidesand oligonucleotide compositions that are chirally controlled. Forinstance, in some embodiments, a provided composition containspredetermined levels of one or more individual oligonucleotide types,wherein an oligonucleotide type is defined by: 1) base sequence; 2)pattern of backbone linkages; 3) pattern of backbone chiral centers; and4) pattern of backbone P-modifications. In some embodiments, aparticular oligonucleotide type may be defined by 1A) base identity; 1B)pattern of base modification; 1C) pattern of sugar modification; 2)pattern of backbone linkages; 3) pattern of backbone chiral centers; and4) pattern of backbone P-modifications. In some embodiments,oligonucleotides of the same oligonucleotide type are identical.

In some embodiments, a provided oligonucleotide is a unimer. In someembodiments, a provided oligonucleotide is a P-modification unimer. Insome embodiments, a provided oligonucleotide is a stereounimer. In someembodiments, a provided oligonucleotide is a stereounimer ofconfiguration Rp. In some embodiments, a provided oligonucleotide is astereounimer of configuration Sp.

In some embodiments, a provided oligonucleotide is an altmer. In someembodiments, a provided oligonucleotide is a P-modification altmer. Insome embodiments, a provided oligonucleotide is a stereoaltmer.

In some embodiments, a provided oligonucleotide is a blockmer. In someembodiments, a provided oligonucleotide is a P-modification blockmer. Insome embodiments, a provided oligonucleotide is a stereoblockmer.

In some embodiments, a provided oligonucleotide is a gapmer.

In some embodiments, a provided oligonucleotide is a skipmer.

In some embodiments, a provided oligonucleotide is a hemimer. In someembodiments, a hemimer is an oligonucleotide wherein the 5′-end or the3′-end has a sequence that possesses a structure feature that the restof the oligonucleotide does not have. In some embodiments, the 5′-end orthe 3′-end has or comprises 2 to 20 nucleotides. In some embodiments, astructural feature is a base modification. In some embodiments, astructural feature is a sugar modification. In some embodiments, astructural feature is a P-modification. In some embodiments, astructural feature is stereochemistry of the chiral internucleotidiclinkage. In some embodiments, a structural feature is or comprises abase modification, a sugar modification, a P-modification, orstereochemistry of the chiral internucleotidic linkage, or combinationsthereof. In some embodiments, a hemimer is an oligonucleotide in whicheach sugar moiety of the 5′-end sequence shares a common modification.In some embodiments, a hemimer is an oligonucleotide in which each sugarmoiety of the 3′-end sequence shares a common modification. In someembodiments, a common sugar modification of the 5′ or 3′ end sequence isnot shared by any other sugar moieties in the oligonucleotide. In someembodiments, an example hemimer is an oligonucleotide comprising asequence of substituted or unsubstituted 2′-O-alkyl sugar modifiednucleosides, bicyclic sugar modified nucleosides, β-D-ribonucleosides orβ-D-deoxyribonucleosides (for example 2′-MOE modified nucleosides, andLNA™ or ENA™ bicyclic syugar modified nucleosides) at one terminus and asequence of nucleosides with a different sugar moiety (such as asubstituted or unsubstituted 2′-O-alkyl sugar modified nucleosides,bicyclic sugar modified nucleosides or natural ones) at the otherterminus. In some embodiments, a provided oligonucleotide is acombination of one or more of unimer, altmer, blockmer, gapmer, hemimerand skipmer. In some embodiments, a provided oligonucleotide is acombination of one or more of unimer, altmer, blockmer, gapmer, andskipmer. For instance, in some embodiments, a provided oligonucleotideis both an altmer and a gapmer. In some embodiments, a providednucleotide is both a gapmer and a skipmer. One of skill in the chemicaland synthetic arts will recognize that numerous other combinations ofpatterns are available and are limited only by the commercialavailability and/or synthetic accessibility of constituent partsrequired to synthesize a provided oligonucleotide in accordance withmethods of the present disclosure. In some embodiments, a hemimerstructure provides advantageous benefits, as exemplified by FIG. 29. Insome embodiments, provided oligonucleotides are 5′-hemmimers thatcomprises modified sugar moieties in a 5′-end sequence. In someembodiments, provided oligonucleotides are 5′-hemmimers that comprisesmodified 2′-sugar moieties in a 5′-end sequence.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleotides. In some embodiments, a providedoligonucleotide comprises one or more modified nucleotides. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted nucleosides. In some embodiments, a provided oligonucleotidecomprises one or more modified nucleosides. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedLNAs.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted nucleobases. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted naturalnucleobases. In some embodiments, a provided oligonucleotide comprisesone or more optionally substituted modified nucleobases. In someembodiments, a provided oligonucleotide comprises one or more5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or5-carboxylcytosine. In some embodiments, a provided oligonucleotidecomprises one or more 5-methylcytidine.

In some embodiments, a provided oligonucleotide comprises one or moreoptionally substituted sugars. In some embodiments, a providedoligonucleotide comprises one or more optionally substituted sugarsfound in naturally occurring DNA and RNA. In some embodiments, aprovided oligonucleotide comprises one or more optionally substitutedribose or deoxyribose. In some embodiments, a provided oligonucleotidecomprises one or more optionally substituted ribose or deoxyribose,wherein one or more hydroxyl groups of the ribose or deoxyribose moietyis optionally and independently replaced by halogen, R′, —N(R′)₂, —OR′,or —SR′, wherein each R′ is independently as defined above and describedherein. In some embodiments, a provided oligonucleotide comprises one ormore optionally substituted deoxyribose, wherein the 2′ position of thedeoxyribose is optionally and independently substituted with halogen,R′, —N(R′)₂, —OR′, or —SR′, wherein each R′ is independently as definedabove and described herein. In some embodiments, a providedoligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with halogen. In some embodiments, aprovided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with one or more —F. halogen. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently as defined above and described herein. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ aliphatic. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OR′, wherein each R′ isindependently an optionally substituted C₁-C₆ alkyl. In someembodiments, a provided oligonucleotide comprises one or more optionallysubstituted deoxyribose, wherein the 2′ position of the deoxyribose isoptionally and independently substituted with —OMe. In some embodiments,a provided oligonucleotide comprises one or more optionally substituteddeoxyribose, wherein the 2′ position of the deoxyribose is optionallyand independently substituted with —O-methoxyethyl.

In some embodiments, a provided oligonucleotide is single-strandedoligonucleotide.

In some embodiments, a provided oligonucleotide is a hybridizedoligonucleotide strand. In certain embodiments, a providedoligonucleotide is a partially hydridized oligonucleotide strand. Incertain embodiments, a provided oligonucleotide is a completelyhydridized oligonucleotide strand. In certain embodiments, a providedoligonucleotide is a double-stranded oligonucleotide. In certainembodiments, a provided oligonucleotide is a triple-strandedoligonucleotide (e.g., a triplex).

In some embodiments, a provided oligonucleotide is chimeric. Forexample, in some embodiments, a provided oligonucleotide is DNA-RNAchimera, DNA-LNA chimera, etc.

In some embodiments, any one of the structures comprising anoligonucleotide depicted in WO2012/030683 can be modified in accordancewith methods of the present disclosure to provide chirally controlledvariants thereof. For example, in some embodiments the chirallycontrolled variants comprise a stereochemical modification at any one ormore of the linkage phosphorus and/or a P-modification at any one ormore of the linkage phosphorus. For example, in some embodiments, aparticular nucleotide unit of an oligonucleotide of WO2012/030683 ispreselected to be stereochemically modified at the linkage phosphorus ofthat nucleotide unit and/or P-modified at the linkage phosphorus of thatnucleotide unit. In some embodiments, a chirally controlledoligonucleotide is of any one of the structures depicted in FIGS. 26-30.In some embodiments, a chirally controlled oligonucleotide is a variant(e.g., modified version) of any one of the structures depicted in FIGS.26-30. The related disclosure of WO2012/030683 is herein incorporated byreference in its entirety.

In some embodiments, a provided oligonucleotide is a therapeutic agent.

In some embodiments, a provided oligonucleotide is an antisenseoligonucleotide.

In some embodiments, a provided oligonucleotide is an antigeneoligonucleotide.

In some embodiments, a provided oligonucleotide is a decoyoligonucleotide.

In some embodiments, a provided oligonucleotide is part of a DNAvaccine.

In some embodiments, a provided oligonucleotide is an immunomodulatoryoligonucleotide, e.g., immunostimulatory oligonucleotide andimmunoinhibitory oligonucleotide.

In some embodiments, a provided oligonucleotide is an adjuvant.

In some embodiments, a provided oligonucleotide is an aptamer.

In some embodiments, a provided oligonucleotide is a ribozyme.

In some embodiments, a provided oligonucleotide is a deoxyribozyme(DNAzymes or DNA enzymes).

In some embodiments, a provided oligonucleotide is an siRNA.

In some embodiments, a provided oligonucleotide is a microRNA, or miRNA.

In some embodiments, a provided oligonucleotide is a ncRNA (non-codingRNAs), including a long non-coding RNA (lncRNA) and a small non-codingRNA, such as piwi-interacting RNA (piRNA).

In some embodiments, a provided oligonucleotide is complementary to astructural RNA, e.g., tRNA.

In some embodiments, a provided oligonucleotide is a nucleic acidanalog, e.g., GNA, LNA, PNA, TNA, GNA, ANA, FANA, CeNA, HNA, UNA, ZNA,or Morpholino. In some embodiments, a provided oligonucleotide is anucleic acid analog, e.g., GNA, LNA, PNA, TNA and Morpholino.

In some embodiments, a provided oligonucleotide is a P-modified prodrug.

In some embodiments, a provided oligonucleotide is a primer. In someembodiments, a primers is for use in polymerase-based chain reactions(i.e., PCR) to amplify nucleic acids. In some embodiments, a primer isfor use in any known variations of PCR, such as reverse transcriptionPCR (RT-PCR) and real-time PCR.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to modulate RNase H activation. For example, in someembodiments, RNase H activation is modulated by the presence ofstereocontrolled phosphorothioate nucleic acid analogs, with naturalDNA/RNA being more or equally susceptible than the Rp stereoisomer,which in turn is more susceptible than the corresponding Spstereoisomer.

In some embodiments, a provided oligonucleotide is characterized ashaving the ability to indirectly or directly increase or decreaseactivity of a protein or inhibition or promotion of the expression of aprotein. In some embodiments, a provided oligonucleotide ischaracterized in that it is useful in the control of cell proliferation,viral replication, and/or any other cell signaling process.

In some embodiments, a provided oligonucleotide is from about 2 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 2 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 2 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 4 to about200 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 180 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about160 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 140 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about120 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 100 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about90 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 80 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about70 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 60 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about50 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 40 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about30 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 29 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about28 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 27 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about26 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 25 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about24 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 23 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about22 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 4 to about 21 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 4 to about20 nucleotide units in length.

In some embodiments, a provided oligonucleotide is from about 5 to about10 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 10 to about 30 nucleotide units in length.In some embodiments, a provided oligonucleotide is from about 15 toabout 25 nucleotide units in length. In some embodiments, a providedoligonucleotide is from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide units in length.

In some embodiments, an oligonucleotide is at least 2 nucleotide unitsin length. In some embodiments, an oligonucleotide is at least 3nucleotide units in length. In some embodiments, an oligonucleotide isat least 4 nucleotide units in length. In some embodiments, anoligonucleotide is at least 5 nucleotide units in length. In someembodiments, an oligonucleotide is at least 6 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 7 nucleotideunits in length. In some embodiments, an oligonucleotide is at least 8nucleotide units in length. In some embodiments, an oligonucleotide isat least 9 nucleotide units in length. In some embodiments, anoligonucleotide is at least 10 nucleotide units in length. In someembodiments, an oligonucleotide is at least 11 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 12nucleotide units in length. In some embodiments, an oligonucleotide isat least 13 nucleotide units in length. In some embodiments, anoligonucleotide is at least 14 nucleotide units in length. In someembodiments, an oligonucleotide is at least 15 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 16nucleotide units in length. In some embodiments, an oligonucleotide isat least 17 nucleotide units in length. In some embodiments, anoligonucleotide is at least 18 nucleotide units in length. In someembodiments, an oligonucleotide is at least 19 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 20nucleotide units in length. In some embodiments, an oligonucleotide isat least 21 nucleotide units in length. In some embodiments, anoligonucleotide is at least 22 nucleotide units in length. In someembodiments, an oligonucleotide is at least 23 nucleotide units inlength. In some embodiments, an oligonucleotide is at least 24nucleotide units in length. In some embodiments, an oligonucleotide isat least 25 nucleotide units in length. In some other embodiments, anoligonucleotide is at least 30 nucleotide units in length. In some otherembodiments, an oligonucleotide is a duplex of complementary strands ofat least 18 nucleotide units in length. In some other embodiments, anoligonucleotide is a duplex of complementary strands of at least 21nucleotide units in length.

In some embodiments, the 5′-end and/or the 3′-end of a providedoligonucleotide is modified. In some embodiments, the 5′-end and/or the3′-end of a provided oligonucleotide is modified with a terminal capmoiety. Example such modifications, including terminal cap moieties areextensively described herein and in the art, for example but not limitedto those described in US Patent Application Publication US2009/0023675A1.

In some embodiments, oligonucleotides of an oligonucleotide typecharacterized by 1) a common base sequence and length, 2) a commonpattern of backbone linkages, and 3) a common pattern of backbone chiralcenters, have the same chemical structure. For example, they have thesame base sequence, the same pattern of nucleoside modifications, thesame pattern of backbone linkages (i.e., pattern of internucleotidiclinkage types, for example, phosphate, phosphorothioate, etc), the samepattern of backbone chiral centers (i.e. pattern of linkage phosphorusstereochemistry (Rp/Sp)), and the same pattern of backbone phosphorusmodifications (e.g., pattern of “-XLR¹” groups in formula I).

Example Oligonucleotides and Compositions

In some embodiments, the present disclosure provides oligonucleotidesand/or oligonucleotide compositions that are useful for various purposesknown in the art. In some embodiments, the present disclosure providesoligonucleotide compositions with improved properties, e.g., activities,toxicities, etc. Non-limiting example compositions are listed below:

TABLE 2 Example Oligonucleotide and Compositions WV-459m5C*m5C*G*T*m5C*G*m5C*m5C*m5C*T*T*m5C*A*G*m5C*A*m5C*G*m5C*A (SEQ ID NO: 39) WV-485m5C*Sm5C*SG*ST*Sm5C*SG*Sm5C*Sm5C*Sm5C*ST*ST*Sm5C*RA*SG*Sm5C*SA*Sm5C*SG*Sm5C* SA (SEQ ID NO: 40) WV-458m5Ceo*m5Ceo*Geo*Teo*m5Ceo*G*m5C*m5C*m5C*T*T*m5C*A*G*m5C*Aeo*m5Ceo*Geo*m5Ceo*Aeo (SEQ ID NO: 41) WV-486m5Ceo*Sm5Ceo*SGeo*STeo*Sm5Ceo*SG*Sm5C*Sm5C*Sm5C*ST*ST*Sm5C*SA*SG*Sm5C*SAeo*Sm5Ceo*SGeo*Sm5Ceo*SAeo (SEQ ID NO: 42) WV-487m5Ceo*Sm5Ceo*SGeo*STeo*Sm5Ceo*SG*Sm5C*Sm5C*Sm5C*ST*ST*Sm5C*RA*SG*Sm5C*SAeo*Sm5Ceo*SGeo*Sm5Ceo*SAeo (SEQ ID NO: 43) WV-488m5Ceo*Rm5Ceo*RGeo*RTeo*Rm5Ceo*RG*Sm5C*Sm5C*Sm5C*ST*ST*Sm5C*RA*SG*Sm5C*SAeo*Rm5Ceo*RGeo*Rm5Ceo*RAeo (SEQ ID NO: 44) ONT-83Geo*Teo*m5Ceo*m5Ceo*m5Ceo*T*G*A*A*G*A*T*G* T*m5C*Aeo*Aeo*Teo*Geo*m5Ceo(SEQ ID NO: 45) ONT-82 Geo*RTeo*Rm5Ceo*Rm5Ceo*Rm5Ceo*RT*RG*RA*RA*RG*RA*RT*RG*RT*Rm5C*RAeo*RAeo*RTeo*RGeo*  Rm5Ceo (SEQ ID NO: 46) ONT-84Geo*STeo*Sm5Ceo*Sm5Ceo*Sm5Ceo*ST*SG*SA*SA*SG*SA*ST*SG*ST*Sm5C*SAeo*SAeo*STeo*SGeo* Sm5Ceo (SEQ ID NO: 47) ONT-85Geo*RTeo*Rm5Ceo*Rm5Ceo*Rm5Ceo*RT*SG*SA*SA*SG*SA*ST*SG*ST*Sm5C*SAeo*RAeo*RTeo* RGeo*Rm5Ceo (SEQ ID NO: 48)ONT-86 Geo*STeo*Sm5Ceo*Sm5Ceo*Sm5Ceo*ST*RG*RA*RA*RG*RA*RT*RG*RT*Rm5C*RAeo*SAeo*STeo*SGeo*   Sm5Ceo (SEQ ID NO: 49) WV-917mG*mG*mC*mA*mC*A*A*G*G*G*C*A*C*A*G*mA* mC*mU*mU*mC (SEQ ID NO: 50)WV-1085 mG*SmG*SmC*SmA*SmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmA*SmC*SmU*SmU*SmC (SEQ ID NO: 51) WV-1086mG*RmG*RmC*RmA*RmC*SA*SA*SG*SG*SG*SC*SA* SC*RA*SG*SmA*RmC*RmU*RmU*RmC(SEQ ID NO: 52) WV-1087 mGmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmUmC (SEQ ID NO: 53) WV-1091mG*RmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmU*RmC (SEQ ID NO: 54) WV-1092mG*SmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA*SG*SmAmCmUmU*SmC (SEQ ID NO: 55) WV-1510G*SmGmCmAmC*SA*SA*SG*SG*SG*SC*SA*SC*RA* SG*SmAmCmUmU*SC (SEQ ID NO: 56)WV-1511 G*mGmCmAmC*A*A*G*G*G*C*A*C*A*G*mAmCmUmU* C (SEQ ID NO: 57)WV-1497 mG*mGmCmAmC*A*A*G*G*G*C*A*C*A*G* mAmCmUmU*mC (SEQ ID NO: 58)WV-1655 Geo*Geom5CeoAeom5Ceo*A*A*G*G*G*C*A*C*A*G*Aeom5CeoTeoTeo*m5Ceo (SEQ ID NO: 59)

TABLE 3 Brief Description of Example Oligonucleotides and Compositions.WV-459 All DNA, each cytidine is 5-methylated, Stereorandom WV-485 AllDNA, each cytidine is 5-methylated, Stereopure, One Rp in DNA WV-4585-10-5 (2′-MOE-DNA-2′-MOE) Gapmer, each cytidine is 5- methylated,Stereorandom WV-486 5-10-5 (2′-MOE-DNA-2′-MOE) Gapmer, each cytidine is5- methylated, Stereopure WV-487 5-10-5 (2′-MOE-DNA-2′-MOE) Gapmer, eachcytidine is 5- methylated, Stereopure, One Rp in DNA WV-488 5-10-5(2′-MOE-DNA-2′-MOE) Gapmer, each cytidine is 5- methylated, Stereopure,One Rp in DNA and Rp wings ONT-83 5-10-5 (2′-MOE-DNA-2′-MOE) Gapmer,each cytidine is 5- methylated, Stereorandom ONT-82 5-10-5(2′-MOE-DNA-2′-MOE) Gapmer, each cytidine is 5- methylated, StereopureONT-84 5-10-5 (2′-MOE-DNA-2′-MOE) Gapmer, each cytidine is 5-methylated, Stereopure ONT-85 5-10-5 (2′-MOE-DNA-2′-MOE) Gapmer, eachcytidine is 5- methylated, Rp wings ONT-86 5-10-5 (2′-MOE-DNA-2′-MOE)Gapmer, each cytidine is 5- methylated, Sp wings WV-917 5-10-5(2′-OMe-DNA-2′-OMe), Gapmer, Stereorandom WV-1085 5-10-5(2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp in DNA WV-1086 5-10-5(2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp in DNA and Rp wingsWV-1087 5-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp in DNA, POwings WV-1091 5-10-5 (2′-OMe-DNA-2′-OMe) Gapmer, Stereopure, One Rp inDNA, PO wings with One Rp on each end WV-1092 5-10-5 (2′-OMe-DNA-2′-OMe)Gapmer, Stereopure, One Rp in DNA, PO wings with One Sp on each endWV-1510 1-4-10-4-1 (DNA-2′-OMe-DNA-2′-OMe-DNA) Gapmer, Stereopure, OneRp in DNA, PO wings with One Sp on each end WV-1511 1-4-10-4-1(DNA-2′-OMe-DNA-2′-OMe-DNA) Gapmer, Stereorandom WV-1497 5-10-5(2′-OMe-DNA-2′-OMe) Gapmer, Stereorandom, PO wings with One PS on eachend WV-1655 5-10-5 (2′-MOE-DNA-2′-MOE) Gapmer, Stereorandom, PO wingswith One PS on each end

In some embodiments, * only represents a stereorandom phosphorothioatelinkage; *S represents an Sp phosphorothioate linkage; *R represents anRp phosphorothioate linkage; all non-labeled linkage is a naturalphosphate linkage; m preceding a base represents 2′-OMe; eo following abase represents 2′-MOE;

In some embodiments, a provided oligonucleotide composition is achirally controlled oligonucleotide composition of an oligonucleotidetype listed in Table 2. In some embodiments, a provided composition isof WV-1092. In some embodiments, a first plurality of oligonucleotidesis a plurality of an oligonucleotide in Table 2. In some embodiments, afirst plurality of oligonucleotides is a plurality of WV-1092.

The present disclosure provides compositions comprising or consisting ofa plurality of provided oligonucleotides (e.g., chirally controlledoligonucleotide compositions). In some embodiments, all such providedoligonucleotides are of the same type, i.e., all have the same basesequence, pattern of backbone linkages (i.e., pattern ofinternucleotidic linkage types, for example, phosphate,phosphorothioate, etc), pattern of backbone chiral centers (i.e. patternof linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbonephosphorus modifications (e.g., pattern of “-XLR¹” groups in formula I).In some embodiments, all oligonucleotides of the same type areidentical. In many embodiments, however, provided compositions comprisea plurality of oligonucleotides types, typically in pre-determinedrelative amounts.

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of one or more providedoligonucleotide types. One of skill in the chemical and medicinal artswill recognize that the selection and amount of each of the one or moretypes of provided oligonucleotides in a provided composition will dependon the intended use of that composition. That is to say, one of skill inthe relevant arts would design a provided chirally controlledoligonucleotide composition such that the amounts and types of providedoligonucleotides contained therein cause the composition as a whole tohave certain desirable characteristics (e.g., biologically desirable,therapeutically desirable, etc.).

In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of two or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of threeor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof four or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of five or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of six or more provided oligonucleotide types.In some embodiments, a provided chirally controlled oligonucleotidecomposition comprises a combination of seven or more providedoligonucleotide types. In some embodiments, a provided chirallycontrolled oligonucleotide composition comprises a combination of eightor more provided oligonucleotide types. In some embodiments, a providedchirally controlled oligonucleotide composition comprises a combinationof nine or more provided oligonucleotide types. In some embodiments, aprovided chirally controlled oligonucleotide composition comprises acombination of ten or more provided oligonucleotide types. In someembodiments, a provided chirally controlled oligonucleotide compositioncomprises a combination of fifteen or more provided oligonucleotidetypes.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration and an amount of chirally uniform mipomersen ofthe Sp configuration.

In some embodiments, a provided chirally controlled oligonucleotidecomposition is a combination of an amount of chirally uniform mipomersenof the Rp configuration, an amount of chirally uniform mipomersen of theSp configuration, and an amount of one or more chirally pure mipomersenof a desired diastereomeric form.

In some embodiments, a provided oligonucleotide type is selected fromthose described in WO/2014/012081 and WO/2015/107425, theoligonucleotides, oligonucleotide types, oligonucleotide compositions,and methods thereof of each of which are incorporated herein byreference. In some embodiments, a provided chirally controlledoligonucleotide composition comprises oligonucleotides of anoligonucleotide type selected from those described in WO/2014/012081 andWO/2015/107425.

In some embodiments, provided oligonucleotides comprise base sequence,pattern of backbone linkages, pattern or backbone chiral centers, and/orpattern of chemical modifications (e.g., base modifications, sugarmodifications, etc.) of any oligonucleotide disclosed herein.

Example Methods for Preparing Oligonucleotides and Compositions

Methods for preparing provided oligonucleotides and oligonucleotidecompositions are widely known in the art, including but not limited tothose described in WO/2010/064146, WO/2011/005761, WO/2013/012758,WO/2014/010250, US2013/0178612, WO/2014/012081 and WO/2015/107425, themethods and reagents of each of which is incorporated herein byreference.

Among other things, the present disclosure provides methods for makingchirally controlled oligonucleotides and chirally controlledcompositions comprising one or more specific nucleotide types. In someembodiments, the phrase “oligonucleotide type,” as used herein, definesan oligonucleotide that has a particular base sequence, pattern ofbackbone linkages, pattern of backbone chiral centers, and pattern ofbackbone phosphorus modifications (e.g., “—XLR¹” groups).Oligonucleotides of a common designated “type” are structurallyidentical to one another with respect to base sequence, pattern ofbackbone linkages, pattern of backbone chiral centers, and pattern ofbackbone phosphorus modifications. In some embodiments, oligonucleotidesof an oligonucleotide type are identical.

In some embodiments, a provided chirally controlled oligonucleotide inthe disclosure has properties different from those of the correspondingstereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has lipophilicity different from that of thestereorandom oligonucleotide mixture. In some embodiments, a chirallycontrolled oligonucleotide has different retention time on HPLC. In someembodiments, a chirally controlled oligonucleotide may have a peakretention time significantly different from that of the correspondingstereorandom oligonucleotide mixture. During oligonucleotidepurification using HPLC as generally practiced in the art, certainchirally controlled oligonucleotides will be largely if not totallylost. During oligonucleotide purification using HPLC as generallypracticed in the art, certain chirally controlled oligonucleotides willbe largely if not totally lost. One of the consequences is that certaindiastereomers of a stereorandom oligonucleotide mixture (certainchirally controlled oligonucleotides) are not tested in assays. Anotherconsequence is that from batches to batches, due to the inevitableinstrumental and human errors, the supposedly “pure” stereorandomoligonucleotide will have inconsistent compositions in thatdiastereomers in the composition, and their relative and absoluteamounts, are different from batches to batches. The chirally controlledoligonucleotide and chirally controlled oligonucleotide compositionprovided in this disclosure overcome such problems, as a chirallycontrolled oligonucleotide is synthesized in a chirally controlledfashion as a single diastereomer, and a chirally controlledoligonucleotide composition comprise predetermined levels of one or moreindividual oligonucleotide types.

One of skill in the chemical and synthetic arts will appreciate thatsynthetic methods of the present disclosure provide for a degree ofcontrol during each step of the synthesis of a provided oligonucleotidesuch that each nucleotide unit of the oligonucleotide can be designedand/or selected in advance to have a particular stereochemistry at thelinkage phosphorus and/or a particular modification at the linkagephosphorus, and/or a particular base, and/or a particular sugar. In someembodiments, a provided oligonucleotide is designed and/or selected inadvance to have a particular combination of stereocenters at the linkagephosphorus of the internucleotidic linkage.

In some embodiments, a provided oligonucleotide made using methods ofthe present disclosure is designed and/or determined to have aparticular combination of linkage phosphorus modifications. In someembodiments, a provided oligonucleotide made using methods of thepresent disclosure is designed and/or determined to have a particularcombination of bases. In some embodiments, a provided oligonucleotidemade using methods of the present disclosure is designed and/ordetermined to have a particular combination of sugars. In someembodiments, a provided oligonucleotide made using methods of thepresent disclosure is designed and/or determined to have a particularcombination of one or more of the above structural characteristics.

Methods of the present disclosure exhibit a high degree of chiralcontrol. For instance, methods of the present disclosure facilitatecontrol of the stereochemical configuration of every single linkagephosphorus within a provided oligonucleotide. In some embodiments,methods of the present disclosure provide an oligonucleotide comprisingone or more modified internucleotidic linkages independently having thestructure of formula I.

In some embodiments, methods of the present disclosure provide anoligonucleotide which is a mipomersen unimer. In some embodiments,methods of the present disclosure provide an oligonucleotide which is amipomersen unimer of configuration Rp. In some embodiments, methods ofthe present disclosure provide an oligonucleotide which is a mipomersenunimer of configuration Sp.

In some embodiments, methods of the present disclosure provide achirally controlled oligonucleotide composition, i.e., anoligonucleotide composition that contains predetermined levels ofindividual oligonucleotide types. In some embodiments a chirallycontrolled oligonucleotide composition comprises one oligonucleotidetype. In some embodiments, a chirally controlled oligonucleotidecomposition comprises more than one oligonucleotide type. In someembodiments, a chirally controlled oligonucleotide composition comprisesa plurality of oligonucleotide types. Example chirally controlledoligonucleotide compositions made in accordance with the presentdisclosure are described herein.

In some embodiments, methods of the present disclosure provide chirallypure mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent disclosure provide compositions of mipomersen wherein mipomersenexists in the composition in the form of a single diastereomer withrespect to the configuration of the linkage phosphorus.

In some embodiments, methods of the present disclosure provide chirallyuniform mipomersen compositions with respect to the configuration of thelinkage phosphorus. That is to say, in some embodiments, methods of thepresent disclosure provide compositions of mipomersen in which allnucleotide units therein have the same stereochemistry with respect tothe configuration of the linkage phosphorus, e.g., all nucleotide unitsare of the Rp configuration at the linkage phosphorus or all nucleotideunits are of the Sp configuration at the linkage phosphorus.

In some embodiments, a provided chirally controlled oligonucleotide isover 50% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 55% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 60% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 65% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 70% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 75% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 80% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 85% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 90% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 91% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 92% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 93% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 94% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 95% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 96% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 97% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 98% pure. In some embodiments, a providedchirally controlled oligonucleotide is over about 99% pure. In someembodiments, a provided chirally controlled oligonucleotide is overabout 99.5% pure. In some embodiments, a provided chirally controlledoligonucleotide is over about 99.6% pure. In some embodiments, aprovided chirally controlled oligonucleotide is over about 99.7% pure.In some embodiments, a provided chirally controlled oligonucleotide isover about 99.8% pure. In some embodiments, a provided chirallycontrolled oligonucleotide is over about 99.9% pure. In someembodiments, a provided chirally controlled oligonucleotide is over atleast about 99% pure.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise a single oligonucleotide type. Incertain embodiments, such compositions are about 50% diastereomericallypure. In some embodiments, such compositions are about 50%diastereomerically pure. In some embodiments, such compositions areabout 50% diastereomerically pure. In some embodiments, suchcompositions are about 55% diastereomerically pure. In some embodiments,such compositions are about 60% diastereomerically pure. In someembodiments, such compositions are about 65% diastereomerically pure. Insome embodiments, such compositions are about 70% diastereomericallypure. In some embodiments, such compositions are about 75%diastereomerically pure. In some embodiments, such compositions areabout 80% diastereomerically pure. In some embodiments, suchcompositions are about 85% diastereomerically pure. In some embodiments,such compositions are about 90% diastereomerically pure. In someembodiments, such compositions are about 91% diastereomerically pure. Insome embodiments, such compositions are about 92% diastereomericallypure. In some embodiments, such compositions are about 93%diastereomerically pure. In some embodiments, such compositions areabout 94% diastereomerically pure. In some embodiments, suchcompositions are about 95% diastereomerically pure. In some embodiments,such compositions are about 96% diastereomerically pure. In someembodiments, such compositions are about 97% diastereomerically pure. Insome embodiments, such compositions are about 98% diastereomericallypure. In some embodiments, such compositions are about 99%diastereomerically pure. In some embodiments, such compositions areabout 99.5% diastereomerically pure. In some embodiments, suchcompositions are about 99.6% diastereomerically pure. In someembodiments, such compositions are about 99.7% diastereomerically pure.In some embodiments, such compositions are about 99.8%diastereomerically pure. In some embodiments, such compositions areabout 99.9% diastereomerically pure. In some embodiments, suchcompositions are at least about 99% diastereomerically pure.

Among other things, the present disclosure recognizes the challenge ofstereoselective (rather than stereorandom or racemic) preparation ofoligonucleotides. Among other things, the present disclosure providesmethods and reagents for stereoselective preparation of oligonucleotidescomprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10)internucleotidic linkages, and particularly for oligonucleotidescomprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiralinternucleotidic linkages. In some embodiments, in a stereorandom orracemic preparation of oligonucleotides, at least one chiralinternucleotidic linkage is formed with less than 90:10, 95:5, 96:4,97:3, or 98:2 diastereoselectivity. In some embodiments, for astereoselective or chirally controlled preparation of oligonucleotides,each chiral internucleotidic linkage is formed with greater than 90:10,95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, fora stereoselective or chirally controlled preparation ofoligonucleotides, each chiral internucleotidic linkage is formed withgreater than 95:5 diastereoselectivity. In some embodiments, for astereoselective or chirally controlled preparation of oligonucleotides,each chiral internucleotidic linkage is formed with greater than 96:4diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 97:3diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 98:2diastereoselectivity. In some embodiments, for a stereoselective orchirally controlled preparation of oligonucleotides, each chiralinternucleotidic linkage is formed with greater than 99:1diastereoselectivity. In some embodiments, diastereoselectivity of achiral internucleotidic linkage in an oligonucleotide may be measuredthrough a model reaction, e.g. formation of a dimer under essentiallythe same or comparable conditions wherein the dimer has the sameinternucleotidic linkage as the chiral internucleotidic linkage, the5′-nucleoside of the dimer is the same as the nucleoside to the 5′-endof the chiral internucleotidic linkage, and the 3′-nucleoside of thedimer is the same as the nucleoside to the 3′-end of the chiralinternucleotidic linkage.

In some embodiments, a chirally controlled oligonucleotide compositionis a composition designed to comprise multiple oligonucleotide types. Insome embodiments, methods of the present disclosure allow for thegeneration of a library of chirally controlled oligonucleotides suchthat a pre-selected amount of any one or more chirally controlledoligonucleotide types can be mixed with any one or more other chirallycontrolled oligonucleotide types to create a chirally controlledoligonucleotide composition. In some embodiments, the pre-selectedamount of an oligonucleotide type is a composition having any one of theabove-described diastereomeric purities.

In some embodiments, the present disclosure provides methods for makinga chirally controlled oligonucleotide comprising steps of:

(1) coupling;

(2) capping;

(3) modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved.

When describing the provided methods, the word “cycle” has its ordinarymeaning as understood by a person of ordinary skill in the art. In someembodiments, one round of steps (1)-(4) is referred to as a cycle.

In some embodiments, the present disclosure provides methods for makingchirally controlled oligonucleotide compositions, comprising steps of:

(a) providing an amount of a first chirally controlled oligonucleotide;and

(b) optionally providing an amount of one or more additional chirallycontrolled oligonucleotides.

In some embodiments, a first chirally controlled oligonucleotide is anoligonucleotide type, as described herein. In some embodiments, a one ormore additional chirally controlled oligonucleotide is a one or moreoligonucleotide type, as described herein.

One of skill in the relevant chemical and synthetic arts will recognizethe degree of versatility and control over structural variation andstereochemical configuration of a provided oligonucleotide whensynthesized using methods of the present disclosure. For instance, aftera first cycle is complete, a subsequent cycle can be performed using anucleotide unit individually selected for that subsequent cycle which,in some embodiments, comprises a nucleobase and/or a sugar that isdifferent from the first cycle nucleobase and/or sugar. Likewise, thechiral auxiliary used in the coupling step of the subsequent cycle canbe different from the chiral auxiliary used in the first cycle, suchthat the second cycle generates a phosphorus linkage of a differentstereochemical configuration. In some embodiments, the stereochemistryof the linkage phosphorus in the newly formed internucleotidic linkageis controlled by using stereochemically pure phosphoramidites.Additionally, the modification reagent used in the modifying step of asubsequent cycle can be different from the modification reagent used inthe first or former cycle. The cumulative effect of this iterativeassembly approach is such that each component of a providedoligonucleotide can be structurally and configurationally tailored to ahigh degree. An additional advantage to this approach is that the stepof capping minimizes the formation of “n−1” impurities that wouldotherwise make isolation of a provided oligonucleotide extremelychallenging, and especially oligonucleotides of longer lengths.

In some embodiments, an example cycle of the method for making chirallycontrolled oligonucleotides is illustrated in example schemes describedin the present disclosure. In some embodiments, an example cycle of themethod for making chirally controlled oligonucleotides is illustrated inScheme I. In some embodiments,

represents the solid support, and optionally a portion of the growingchirally controlled oligonucleotide attached to the solid support. Thechiral auxiliary exemplified has the structure of formula 3-I:

which is further described below. “Cap” is any chemical moietyintroduced to the nitrogen atom by the capping step, and in someembodiments, is an amino protecting group. One of ordinary skill in theart understands that in the first cycle, there may be only onenucleoside attached to the solid support when started, and cycle exitcan be performed optionally before deblocking. As understood by a personof skill in the art, B^(PRO) is a protected base used in oligonucleotidesynthesis. Each step of the above-depicted cycle of Scheme I isdescribed further below.

Synthesis on Solid Support

In some embodiments, the synthesis of a provided oligonucleotide isperformed on solid phase. In some embodiments, reactive groups presenton a solid support are protected. In some embodiments, reactive groupspresent on a solid support are unprotected. During oligonucleotidesynthesis a solid support is treated with various reagents in severalsynthesis cycles to achieve the stepwise elongation of a growingoligonucleotide chain with individual nucleotide units. The nucleosideunit at the end of the chain which is directly linked to the solidsupport is termed “the first nucleoside” as used herein. A firstnucleoside is bound to a solid support via a linker moiety, i.e. adiradical with covalent bonds between either of a CPG, a polymer orother solid support and a nucleoside. The linker stays intact during thesynthesis cycles performed to assemble the oligonucleotide chain and iscleaved after the chain assembly to liberate the oligonucleotide fromthe support.

Solid supports for solid-phase nucleic acid synthesis include thesupports described in, e.g., U.S. Pat. Nos. 4,659,774, 5,141,813,4,458,066; Caruthers U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707,4,668,777, 4,973,679, and 5,132,418; Andrus et al. U.S. Pat. Nos.5,047,524, 5,262,530; and Koster U.S. Pat. No. 4,725,677 (reissued asRE34,069). In some embodiments, a solid phase is an organic polymersupport. In some embodiments, a solid phase is an inorganic polymersupport. In some embodiments, an organic polymer support is polystyrene,aminomethyl polystyrene, a polyethylene glycol-polystyrene graftcopolymer, polyacrylamide, polymethacrylate, polyvinylalcohol, highlycross-linked polymer (HCP), or other synthetic polymers, carbohydratessuch as cellulose and starch or other polymeric carbohydrates, or otherorganic polymers and any copolymers, composite materials or combinationof the above inorganic or organic materials. In some embodiments, aninorganic polymer support is silica, alumina, controlled polyglass(CPG), which is a silica-gel support, or aminopropyl CPG. Other usefulsolid supports include fluorous solid supports (see e.g.,WO/2005/070859), long chain alkylamine (LCAA) controlled pore glass(CPG) solid supports (see e.g., S. P. Adams, K. S. Kavka, E. J. Wykes,S. B. Holder and G. R. Galluppi, J. Am. Chem. Soc., 1983, 105, 661-663;G. R. Gough, M. J. Bruden and P. T. Gilham, Tetrahedron Lett., 1981, 22,4177-4180). Membrane supports and polymeric membranes (see e.g.Innovation and Perspectives in Solid Phase Synthesis, Peptides, Proteinsand Nucleic Acids, ch 21 pp 157-162, 1994, Ed. Roger Epton and U.S. Pat.No. 4,923,901) are also useful for the synthesis of nucleic acids. Onceformed, a membrane can be chemically functionalized for use in nucleicacid synthesis. In addition to the attachment of a functional group tothe membrane, the use of a linker or spacer group attached to themembrane is also used in some embodiments to minimize steric hindrancebetween the membrane and the synthesized chain.

Other suitable solid supports include those generally known in the artto be suitable for use in solid phase methodologies, including, forexample, glass sold as Primer™ 200 support, controlled pore glass (CPG),oxalyl-controlled pore glass (see, e.g., Alul, et al., Nucleic AcidsResearch, 1991, 19, 1527), TentaGel Support-an aminopolyethyleneglycolderivatized support (see, e.g., Wright, et al., Tetrahedron Lett., 1993,34, 3373), and Poros-a copolymer of polystyrene/divinylbenzene.

Surface activated polymers have been demonstrated for use in synthesisof natural and modified nucleic acids and proteins on several solidsupports mediums. A solid support material can be any polymer suitablyuniform in porosity, having sufficient amine content, and sufficientflexibility to undergo any attendant manipulations without losingintegrity. Examples of suitable selected materials include nylon,polypropylene, polyester, polytetrafluoroethylene, polystyrene,polycarbonate, and nitrocellulose. Other materials can serve as a solidsupport, depending on the design of the investigator. In considerationof some designs, for example, a coated metal, in particular gold orplatinum can be selected (see e.g., US publication No. 20010055761). Inone embodiment of oligonucleotide synthesis, for example, a nucleosideis anchored to a solid support which is functionalized with hydroxyl oramino residues. Alternatively, a solid support is derivatized to providean acid labile trialkoxytrityl group, such as a trimethoxytrityl group(TMT). Without being bound by theory, it is expected that the presenceof a trialkoxytrityl protecting group will permit initial detritylationunder conditions commonly used on DNA synthesizers. For a faster releaseof oligonucleotide material in solution with aqueous ammonia, adiglycoate linker is optionally introduced onto the support.

In some embodiments, a provided oligonucleotide alternatively issynthesized from the 5′ to 3′ direction. In some embodiments, a nucleicacid is attached to a solid support through its 5′ end of the growingnucleic acid, thereby presenting its 3′ group for reaction, i.e. using5′-nucleoside phosphoramidites or in enzymatic reaction (e.g. ligationand polymerization using nucleoside 5′-triphosphates). When consideringthe 5′ to 3′ synthesis the iterative steps of the present disclosureremain unchanged (i.e. capping and modification on the chiralphosphorus).

Linking Moiety

A linking moiety or linker is optionally used to connect a solid supportto a compound comprising a free nucleophilic moiety. Suitable linkersare known such as short molecules which serve to connect a solid supportto functional groups (e.g., hydroxyl groups) of initial nucleosidesmolecules in solid phase synthetic techniques. In some embodiments, thelinking moiety is a succinamic acid linker, or a succinate linker(—CO—CH₂—CH₂—CO—), or an oxalyl linker (—CO—CO—). In some embodiments,the linking moiety and the nucleoside are bonded together through anester bond. In some embodiments, a linking moiety and a nucleoside arebonded together through an amide bond. In some embodiments, a linkingmoiety connects a nucleoside to another nucleotide or nucleic acid.Suitable linkers are disclosed in, for example, Oligonucleotides AndAnalogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991,Chapter 1 and Solid-Phase Supports for Oligonucleotide Synthesis, Pon,R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28.

A linker moiety is used to connect a compound comprising a freenucleophilic moiety to another nucleoside, nucleotide, or nucleic acid.In some embodiments, a linking moiety is a phosphodiester linkage. Insome embodiments, a linking moiety is an H-phosphonate moiety. In someembodiments, a linking moiety is a modified phosphorus linkage asdescribed herein. In some embodiments, a universal linker (UnyLinker) isused to attached the oligonucleotide to the solid support (Ravikumar etal., Org. Process Res. Dev., 2008, 12 (3), 399-410). In someembodiments, other universal linkers are used (Pon, R. T., Curr. Prot.Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, variousorthogonal linkers (such as disulfide linkers) are used (Pon, R. T.,Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

Among other things, the present disclosure recognizes that a linker canbe chosen or designed to be compatible with a set of reaction conditionsemployed in oligonucleotide synthesis. In some embodiments, to avoiddegradation of oligonucleotides and to avoid desulfurization, auxiliarygroups are selectively removed before de-protection. In someembodiments, DPSE group can selectively be removed by F⁻ ions. In someembodiments, the present disclosure provides linkers that are stableunder a DPSE de-protection condition, e.g., 0.1M TBAF in MeCN, 0.5MHF-Et₃N in THF or MeCN, etc. In some embodiments, a provided linker isthe SP linker. In some embodiments, the present disclosure demonstratesthat the SP linker is stable under a DPSE de-protection condition, e.g.,0.1M TBAF in MeCN, 0.5M HF-Et₃N in THF or MeCN, etc.; they are alsostable, e.g., under anhydrous basic conditions, such as om1M DBU inMeCN.

In some embodiments, an example linker is:

In some embodiments, the succinyl linker, Q-linker or oxalyl linker isnot stable to one or more DPSE-deprotection conditions using F⁻.

General Conditions—Solvents for Synthesis

Syntheses of provided oligonucleotides are generally performed inaprotic organic solvents. In some embodiments, a solvent is a nitrilesolvent such as, e.g., acetonitrile. In some embodiments, a solvent is abasic amine solvent such as, e.g., pyridine. In some embodiments, asolvent is an ethereal solvent such as, e.g., tetrahydrofuran. In someembodiments, a solvent is a halogenated hydrocarbon such as, e.g.,dichloromethane. In some embodiments, a mixture of solvents is used. Incertain embodiments a solvent is a mixture of any one or more of theabove-described classes of solvents.

In some embodiments, when an aprotic organic solvent is not basic, abase is present in the reacting step. In some embodiments where a baseis present, the base is an amine base such as, e.g., pyridine,quinoline, or N,N-dimethylaniline. Example other amine bases includepyrrolidine, piperidine, N-methyl pyrrolidine, pyridine, quinoline,N,N-dimethylaminopyridine (DMAP), or N,N-dimethylaniline.

In some embodiments, a base is other than an amine base.

In some embodiments, an aprotic organic solvent is anhydrous. In someembodiments, an anhydrous aprotic organic solvent is freshly distilled.In some embodiments, a freshly distilled anhydrous aprotic organicsolvent is a basic amine solvent such as, e.g., pyridine. In someembodiments, a freshly distilled anhydrous aprotic organic solvent is anethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, afreshly distilled anhydrous aprotic organic solvent is a nitrile solventsuch as, e.g., acetonitrile.

Chiral Reagent/Chiral Auxiliary

In some embodiments, chiral reagents are used to conferstereoselectivity in the production of chirally controlledolignucleotides. Many different chiral reagents, also referred to bythose of skill in the art and herein as chiral auxiliaries, may be usedin accordance with methods of the present disclosure. Example suchchiral reagents are described herein and in Wada I, II and III,referenced above. In certain embodiments, a chiral reagent is asdescribed by Wada I. In some embodiments, a chiral reagent for use inaccordance with the methods of the present disclosure are of Formula3-I, below:

wherein W¹ and W² are any of —O—, —S—, or -NG⁵-, U₁ and U₃ are carbonatoms which are bonded to U₂ if present, or to each other if r is 0, viaa single, double or triple bond. U2 is —C—, -CG⁸-, -CG⁸G⁸-, -NG⁸-, —N—,—O—, or —S— where r is an integer of 0 to 5 and no more than twoheteroatoms are adjacent. When any one of U₂ is C, a triple bond must beformed between a second instance of U₂, which is C, or to one of U₁ orU₃. Similarly, when any one of U₂ is CG⁸, a double bond is formedbetween a second instance of U₂ which is -CG⁸- or —N—, or to one of U₁or U₃. In some embodiments, —U₁(G³G⁴)-(U₂)_(r)—U₃(G¹G²)— — is-CG³G⁴-CG¹G²-. In some embodiments, —U₁—(U₂)_(r)—U₃— is -CG³=CG¹-. Insome embodiments, —U₁—(U₂)_(r)—U₃— is —C≡C—. In some embodiments,—U₁—(U₂)_(r)—U₃— is -CG³=CG-CG¹G²-. In some embodiments,—U₁—(U₂)_(r)—U₃— is -CG³G⁴-O-CG¹G²-. In some embodiments,—U₁—(U₂)_(r)—U₃— is -CG³G⁴-NG-CG¹G²-. In some embodiments,—U₁—(U₂)_(r)—U₃— is -CG³G⁴-N-CG²-. In some embodiments, —U₁—(U₂)_(r)—U₃—is -CG³G⁴-N═C G⁸-CG¹G²-.

As defined herein, G¹, G², G³, G⁴, G⁵, and G⁸ are independentlyhydrogen, or an optionally substituted group selected from alkyl,aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, andaryl; or two of G¹, G², G³, G⁴, and G⁵ are G⁶ (taken together to form anoptionally substituted, saturated, partially unsaturated or unsaturatedcarbocyclic or heteroatom-containing ring of up to about 20 ring atomswhich is monocyclic or polycyclic, and is fused or unfused). In someembodiments, a ring so formed is substituted by oxo, thioxo, alkyl,alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments,when a ring formed by taking two G⁶ together is substituted, it issubstituted by a moiety which is bulky enough to conferstereoselectivity during the reaction.

In some embodiments, a ring formed by taking two of G⁶ together isoptionally substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl,cyclopentenyl, tetrahydropyranyl, or piperazinyl. In some embodiments, aring formed by taking two of G⁶ together is optionally substitutedcyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl,tetrahydropyranyl, pyrrolidinyl, or piperazinyl.

In some embodiments, G¹ is optionally substituted phenyl. In someembodiments, G¹ is phenyl. In some embodiments, G² is methyl orhydrogen. In some embodiments, G¹ is optionally substituted phenyl andG² is methyl. In some embodiments, G¹ is phenyl and G² is methyl.

In some embodiments, r is 0.

In some embodiments, W¹ is -NG⁵-. In some embodiments, one of G³ and G⁴is taken together with G⁵ to form an optionally substituted pyrrolidinylring. In some embodiments, one of G³ and G⁴ is taken together with G⁵ toform a pyrrolidinyl ring.

In some embodiments, W² is —O—.

In some embodiments, a chiral reagent is a compound of Formula 3-AA:

wherein each variable is independently as defined above and describedherein.

In some embodiments of Formula 3AA, W¹ and W² are independently -NG⁵-,—O—, or —S—; G¹, G², G³, G⁴, and G⁵ are independently hydrogen, or anoptionally substituted group selected from alkyl, aralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, heteroaryl, or aryl; or two of G¹, G²,G³, G⁴, and G⁵ are G⁶ (taken together to form an optionally substitutedsaturated, partially unsaturated or unsaturated carbocyclic orheteroatom-containing ring of up to about 20 ring atoms which ismonocyclic or polycyclic, fused or unfused), and no more than four ofG¹, G², G³, G⁴, and G⁵ are G⁶. Similarly to the compounds of Formula3-I, any of G¹, G², G³, G⁴, or G⁵ are optionally substituted by oxo,thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In someembodiments, such substitution induces stereoselectivity in chirallycontrolled oligonucleotide production.

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of G.

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, a provided chiral reagent has the structure of

In some embodiments, W¹ is -NG⁵, W² is O, each of G¹ and G³ isindependently hydrogen or an optionally substituted group selected fromC₁₋₁₀ aliphatic, heterocyclyl, heteroaryl and aryl, G² is —C(R)₂Si(R)₃,and G⁴ and G⁵ are taken together to form an optionally substitutedsaturated, partially unsaturated or unsaturated heteroatom-containingring of up to about 20 ring atoms which is monocyclic or polycyclic,fused or unfused. In some embodiments, each R is independently hydrogen,or an optionally substituted group selected from C₁-C₆ aliphatic,carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, G²is —C(R)₂Si(R)₃, wherein —C(R)₂— is optionally substituted —CH₂—, andeach R of —Si(R)₃ is independently an optionally substituted groupselected from C₁₋₁₀ aliphatic, heterocyclyl, heteroaryl and aryl. Insome embodiments, at least one R of —Si(R)₃ is independently optionallysubstituted C₁₋₁₀ alkyl. In some embodiments, at least one R of —Si(R)₃is independently optionally substituted phenyl. In some embodiments, oneR of —Si(R)₃ is independently optionally substituted phenyl, and each ofthe other two R is independently optionally substituted C₁₋₁₀ alkyl. Insome embodiments, one R of —Si(R)₃ is independently optionallysubstituted C₁₋₁₀ alkyl, and each of the other two R is independentlyoptionally substituted phenyl. In some embodiments, G² is optionallysubstituted —CH₂Si(Ph)(Me)₂. In some embodiments, G² is optionallysubstituted —CH₂Si(Me)(Ph)₂. In some embodiments, G² is —CH₂Si(Me)(Ph)₂.In some embodiments, G⁴ and G⁵ are taken together to form an optionallysubstituted saturated 5-6 membered ring containing one nitrogen atom (towhich G⁵ is attached). In some embodiments, G⁴ and G⁵ are taken togetherto form an optionally substituted saturated 5-membered ring containingone nitrogen atom. In some embodiments, G¹ is hydrogen. In someembodiments, G³ is hydrogen. In some embodiments, both G¹ and G³ arehydrogen.

In some embodiments, a chiral reagent has one of the following formulae:

In some embodiments, a chiral reagent is an aminoalcohol. In someembodiments, a chiral reagent is an aminothiol. In some embodiments, achiral reagent is an aminophenol. In some embodiments, a chiral reagentis (S)- and (R)-2-methylamino-1-phenylethanol, (1R, 2S)-ephedrine, or(1R, 2S)-2-methylamino-1,2-diphenylethanol.

In some embodiments of the disclosure, a chiral reagent is a compound ofone of the following formulae:

As demonstrated herein, when used for preparing a chiralinternucleotidic linkage, to obtain stereoselectivity generallystereochemically pure chiral reagents are utilized. Among other things,the present disclosure provides stereochemically pure chiral reagents,including those having structures described.

The choice of chiral reagent, for example, the isomer represented byFormula Q or its stereoisomer, Formula R, permits specific control ofchirality at a linkage phosphorus. Thus, either an Rp or Spconfiguration can be selected in each synthetic cycle, permittingcontrol of the overall three dimensional structure of a chirallycontrolled oligonucleotide. In some embodiments, a chirally controlledoligonucleotide has all Rp stereocenters. In some embodiments of thedisclosure, a chirally controlled oligonucleotide has all Spstereocenters. In some embodiments of the disclosure, each linkagephosphorus in the chirally controlled oligonucleotide is independentlyRp or Sp. In some embodiments of the disclosure, each linkage phosphorusin the chirally controlled oligonucleotide is independently Rp or Sp,and at least one is Rp and at least one is Sp. In some embodiments, theselection of Rp and Sp centers is made to confer a specific threedimensional superstructure to a chirally controlled oligonucleotide.Example such selections are described in further detail herein.

In some embodiments, a chiral reagent for use in accordance with thepresent disclosure is selected for its ability to be removed at aparticular step in the above-depicted cycle. For example, in someembodiments it is desirable to remove a chiral reagent during the stepof modifying the linkage phosphorus. In some embodiments, it isdesirable to remove a chiral reagent before the step of modifying thelinkage phosphorus. In some embodiments, it is desirable to remove achiral reagent after the step of modifying the linkage phosphorus. Insome embodiments, it is desirable to remove a chiral reagent after afirst coupling step has occurred but before a second coupling step hasoccurred, such that a chiral reagent is not present on the growingoligonucleotide during the second coupling (and likewise for additionalsubsequent coupling steps). In some embodiments, a chiral reagent isremoved during the “deblock” reaction that occurs after modification ofthe linkage phosphorus but before a subsequent cycle begins. Examplemethods and reagents for removal are described herein.

In some embodiments, removal of chiral auxiliary is achieved whenperforming the modification and/or deblocking step, as illustrated inScheme I. It can be beneficial to combine chiral auxiliary removaltogether with other transformations, such as modification anddeblocking. A person of ordinary skill in the art would appreciate thatthe saved steps/transformation could improve the overall efficiency ofsynthesis, for instance, with respect to yield and product purity,especially for longer oligonucleotides. One example wherein the chiralauxiliary is removed during modification and/or deblocking isillustrated in Scheme I.

In some embodiments, a chiral reagent for use in accordance with methodsof the present disclosure is characterized in that it is removable undercertain conditions. For instance, in some embodiments, a chiral reagentis selected for its ability to be removed under acidic conditions. Incertain embodiments, a chiral reagent is selected for its ability to beremoved under mildly acidic conditions. In certain embodiments, a chiralreagent is selected for its ability to be removed by way of an E1elimination reaction (e.g., removal occurs due to the formation of acation intermediate on the chiral reagent under acidic conditions,causing the chiral reagent to cleave from the oligonucleotide). In someembodiments, a chiral reagent is characterized in that it has astructure recognized as being able to accommodate or facilitate an E1elimination reaction. One of skill in the relevant arts will appreciatewhich structures would be envisaged as being prone toward undergoingsuch elimination reactions.

In some embodiments, a chiral reagent is selected for its ability to beremoved with a nucleophile. In some embodiments, a chiral reagent isselected for its ability to be removed with an amine nucleophile. Insome embodiments, a chiral reagent is selected for its ability to beremoved with a nucleophile other than an amine.

In some embodiments, a chiral reagent is selected for its ability to beremoved with a base. In some embodiments, a chiral reagent is selectedfor its ability to be removed with an amine. In some embodiments, achiral reagent is selected for its ability to be removed with a baseother than an amine.

Additional chiral auxiliaries and their use can be found in e.g., Wada I(JP4348077; WO2005/014609; WO2005/092909), Wada II (WO2010/064146), WadaIII (WO2012/039448), Chiral Control (WO2010/064146), etc.

Activation

An achiral H-phosphonate moiety is treated with the first activatingreagent to form the first intermediate. In one embodiment, the firstactivating reagent is added to the reaction mixture during thecondensation step. Use of the first activating reagent is dependent onreaction conditions such as solvents that are used for the reaction.Examples of the first activating reagent are phosgene, trichloromethylchloroformate, bis(trichloromethyl)carbonate (BTC), oxalyl chloride,Ph₃PCl₂, (PhO)₃PCl₂, N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride(BopCl),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP).

The example of achiral H-phosphonate moiety is a compound shown in theabove Scheme. DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene. H⁺DBUmay be, for example, ammonium ion, alkylammonium ion, heteroaromaticiminium ion, or heterocyclic iminium ion, any of which is primary,secondary, tertiary or quaternary, or a monovalent metal ion. Reactingwith Chiral Reagent

After the first activation step, the activated achiral H-phosphonatemoiety reacts with a chiral reagent, which is represented by formula(Z-I) or (Z-I′), to form a chiral intermediate of formula (Z-Va),(Z-Vb), (Z-Va′), or (Z-Vb′).

Stereospecific Condensation Step

A chiral intermediate of Formula Z-Va ((Z-Vb), (Z-Va′), or (Z-Vb′)) istreated with the second activating reagent and a nucleoside to form acondensed intermediate. The nucleoside may be on solid support. Examplesof the second activating reagent are 4,5-dicyanoimidazole (DCI),4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT),benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole(NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole(BTT), 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate(CMPT), N-cyanomethylpiperidinium triflate,N-cyanomethyldimethylammonium triflate. A chiral intermediate of FormulaZ-Va ((Z-Vb), (Z-Va′), or (Z-Vb′)) may be isolated as a monomer.Usually, the chiral intermediate of Z-Va ((Z-Vb), (Z-Va′), or (Z-Vb′))is not isolated and undergoes a reaction in the same pot with anucleoside or modified nucleoside to provide a chiral phosphitecompound, a condensed intermediate. In other embodiments, when themethod is performed via solid phase synthesis, the solid supportcomprising the compound is filtered away from side products, impurities,and/or reagents.

Capping Step

If the final nucleic acid is larger than a dimer, the unreacted —OHmoiety is capped with a blocking group and the chiral auxiliary in thecompound may also be capped with a blocking group to form a cappedcondensed intermediate. If the final nucleic acid is a dimer, then thecapping step is not necessary.

Modifying Step

The compound is modified by reaction with an electrophile. The cappedcondensed intermediate may be executed modifying step. In someembodiments, the modifying step is performed using a sulfurelectrophile, a selenium electrophile or a boronating agent. Examples ofmodifying steps are step of oxidation and sulfurization.

In some embodiments of the method, the sulfur electrophile is a compoundhaving one of the following formulas:

S₈(Formula Z-B), Z^(z1)—S—S—Z^(z2), or Z^(z1)—S—V^(z)—Z^(z2);

wherein Z^(z1) and Z^(z2) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z1) and Z^(z2) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, O, or NR^(f); and R^(f) is hydrogen, alkyl,alkenyl, alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compoundof following Formulae Z-A, Z—B, Z—C, Z-D, Z-E, or Z-F:

In some embodiments, a sulfurization reagent is3-phenyl-1,2,4-dithiazolin-5-one.

In some embodiments, the selenium electrophile is a compound having oneof the following formulae:

Se (Formula Z-G), Z^(z3)—Se—Se—Z^(z4), or Z^(z3)—Se—V^(z)—Z^(z4);

wherein Z^(z3) and Z^(z4) are independently alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, orthiocarbonyl, or Z^(z3) and Z^(z4) are taken together to form a 3 to 8membered alicyclic or heterocyclic ring, which may be substituted orunsubstituted; V^(z) is SO₂, S, O, or NR^(f); and R^(f) is hydrogen,alkyl, alkenyl, alkynyl, or aryl.

In some embodiments, the selenium electrophile is a compound of FormulaZ-G, Z—H, Z—I, Z-J, Z-K, or Z-L.

In some embodiments, the boronating agent isborane-N,N-diisopropylethylamine (BH₃ DIPEA), borane-pyridine (BH₃ Py),borane-2-chloropyridine (BH₃ CPy), borane-aniline (BH₃ An),borane-tetrahydrofiirane (BH₃ THF), or borane-dimethylsulfide (BH₃Me₂S).

In some embodiments, after the modifying step, a chiral auxiliary groupfalls off from the growing oligonucleotide chain. In some embodiments,after the modifying step, a chiral auxiliary group remains connected tothe internucleotidic phosphorus atom.

In some embodiments of the method, the modifying step is an oxidationstep. In some embodiments of the method, the modifying step is anoxidation step using similar conditions as described above in thisapplication. In some embodiments, an oxidation step is as disclosed in,e.g., JP 2010-265304 A and WO2010/064146.

Chain Elongation Cycle and De-Protection Step

The capped condensed intermediate is deblocked to remove the blockinggroup at the 5′-end of the growing nucleic acid chain to provide acompound. The compound is optionally allowed to re-enter the chainelongation cycle to form a condensed intermediate, a capped condensedintermediate, a modified capped condensed intermediate, and a5′-deprotected modified capped intermediate. Following at least oneround of chain elongation cycle, the 5′-deprotected modified cappedintermediate is further deblocked by removal of the chiral auxiliaryligand and other protecting groups for, e.g., nucleobase, modifiednucleobase, sugar and modified sugar protecting groups, to provide anucleic acid. In other embodiments, the nucleoside comprising a 5′-OHmoiety is an intermediate from a previous chain elongation cycle asdescribed herein. In yet other embodiments, the nucleoside comprising a5′-OH moiety is an intermediate obtained from another known nucleic acidsynthetic method. In embodiments where a solid support is used, thephosphorus-atom modified nucleic acid is then cleaved from the solidsupport. In certain embodiments, the nucleic acids is left attached onthe solid support for purification purposes and then cleaved from thesolid support following purification.

In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic method.In yet other embodiments, the nucleoside comprising a 5′-OH moiety is anintermediate obtained from another known nucleic acid synthetic methodas described in this application. In yet other embodiments, thenucleoside comprising a 5′-OH moiety is an intermediate obtained fromanother known nucleic acid synthetic method comprising one or morecycles illustrated in Scheme I. In yet other embodiments, the nucleosidecomprising a 5′-OH moiety is an intermediate obtained from another knownnucleic acid synthetic method comprising one or more cycles illustratedin Scheme I-b, I-c or I-d.

In some embodiments, the present disclosure provides oligonucleotidesynthesis methods that use stable and commercially available materialsas starting materials. In some embodiments, the present disclosureprovides oligonucleotide synthesis methods to produce stereocontrolledphosphorus atom-modified oligonucleotide derivatives using an achiralstarting material.

In some embodiments, the method of the present disclosure does not causedegradations under the de-protection steps. Further the method does notrequire special capping agents to produce phosphorus atom-modifiedoligonucleotide derivatives.

Condensing Reagent

Condensing reagents (C_(R)) useful in accordance with methods of thepresent disclosure are of any one of the following general formulae:

wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, Z⁸, and Z⁹ are independentlyoptionally substituted group selected from alkyl, aminoalkyl,cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl,heteroaryl, alkyloxy, aryloxy, or heteroaryloxy, or wherein any of Z²and Z³, Z⁵ and Z⁶, Z⁷ and Z⁸, Z⁸ and Z⁹, Z⁹ and Z⁷, or Z⁷ and Z⁸ and Z⁹are taken together to form a 3 to 20 membered alicyclic or heterocyclicring; Q⁻ is a counter anion; and LG is a leaving group.

In some embodiments, a counter ion of a condensing reagent C_(R) is Cl⁻,Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, wherein Tf isCF₃SO₂. In some embodiments, a leaving group of a condensing reagentC_(R) is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole,tetrazole, pentafluorobenzene, or 1-hydroxybenzotriazole.

Examples of condensing reagents used in accordance with methods of thepresent disclosure include, but are not limited to, pentafluorobenzoylchloride, carbonyldiimidazole (CDI),1-mesitylenesulfonyl-3-nitrotriazole (MSNT),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCl),benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(PyBOP), N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BopCl),2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), andO-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU),DIPCDI; N,N′-bis(2-oxo-3-oxazolidinyl)phosphinic bromide (BopBr),1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP),3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP), bromotripyrrolidinophosphoniumhexafluorophosphate (PyBrOP);O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU); and tetramethylfluoroformamidinium hexafluorophosphate (TFFH).In certain embodiments, a counter ion of the condensing reagent C_(R) isCl⁻, Br⁻, BF₄ ⁻, PF₆ ⁻, TfO⁻, Tf₂N⁻, AsF₆ ⁻, ClO₄ ⁻, or SbF₆ ⁻, whereinTf is CF₃SO₂.

In some embodiments, a condensing reagent is1-(2,4,6-triisopropylbenzenesulfonyl)-5-(pyridin-2-yl) tetrazolide,pivaloyl chloride, bromotrispyrrolidinophosphonium hexafluorophosphate,N,N′-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl), or2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane. In some embodiment,a condensing reagent is N,N′-bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BopCl). In some embodiments, a condensing reagent is selectedfrom those described in WO/2006/066260).

In some embodiments, a condensing reagent is1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate (MNTP), or3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyNTP):

Selection of Base and Sugar of Nucleoside Coupling Partner

As described herein, nucleoside coupling partners for use in accordancewith methods of the present disclosure can be the same as one another orcan be different from one another. In some embodiments, nucleosidecoupling partners for use in the synthesis of a provided oligonucleotideare of the same structure and/or stereochemical configuration as oneanother. In some embodiments, each nucleoside coupling partner for usein the synthesis of a provided oligonucleotide is not of the samestructure and/or stereochemical configuration as certain othernucleoside coupling partners of the oligonucleotide. Example nucleobasesand sugars for use in accordance with methods of the present disclosureare described herein. One of skill in the relevant chemical andsynthetic arts will recognize that any combination of nucleobases andsugars described herein are contemplated for use in accordance withmethods of the present disclosure.

Coupling Step

Example coupling procedures and chiral reagents and condensing reagentsfor use in accordance with the present disclosure are outlined in, interalia, Wada I (JP4348077; WO2005/014609; WO2005/092909), Wada II(WO2010/064146), Wada III (WO2012/039448), and Chiral Control(WO2010/064146). Chiral nucleoside coupling partners for use inaccordance with the present disclosure are also referred to herein as“Wada amidites.” In some embodiments, a coupling partner has thestructure of

wherein B^(PRO) is a protected nucleobase. In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase. In some embodiments, acoupling partner has the structure of

wherein B^(PRO) is a protected nucleobase, and R¹ is as defined anddescribed herein. In some embodiments, a coupling partner has thestructure of

wherein B^(PRO) is a protected nucleobase, and R¹ is as defined anddescribed herein. In some embodiments, R¹ is optionally substituted C₁₋₆alkyl. In some embodiments, R¹ is Me.

Example chiral phosphoramidites as coupling partner are depicted below:

Additional examples are described in Chiral Control (WO2010/064146).

One of the methods used for synthesizing the coupling partner isdepicted in Scheme II, below.

In some embodiments, the step of coupling comprises reacting a freehydroxyl group of a nucleotide unit of an oligonucleotide with anucleoside coupling partner under suitable conditions to effect thecoupling. In some embodiments, the step of coupling is preceded by astep of deblocking. For instance, in some embodiments, the 5′ hydroxylgroup of the growing oligonucleotide is blocked (i.e., protected) andmust be deblocked in order to subsequently react with a nucleosidecoupling partner.

Once the appropriate hydroxyl group of the growing oligonucleotide hasbeen deblocked, the support is washed and dried in preparation fordelivery of a solution comprising a chiral reagent and a solutioncomprising an activator. In some embodiments, a chiral reagent and anactivator are delivered simultaneously. In some embodiments, co-deliverycomprises delivering an amount of a chiral reagent in solution (e.g., aphosphoramidite solution) and an amount of activator in a solution(e.g., a CMPT solution) in a polar aprotic solvent such as a nitrilesolvent (e.g., acetonitrile).

In some embodiments, the step of coupling provides a crude productcomposition in which the chiral phosphite product is present in adiastereomeric excess of >95%. In some embodiments, the chiral phosphiteproduct is present in a diastereomeric excess of >96%. In someembodiments, the chiral phosphite product is present in a diastereomericexcess of >97%. In some embodiments, the chiral phosphite product ispresent in a diastereomeric excess of >98%. In some embodiments, thechiral phosphite product is present in a diastereomeric excess of >99%.

Capping Step:

Provided methods for making chirally controlled oligonucleotidescomprise a step of capping. In some embodiments, a step of capping is asingle step. In some embodiments, a step of capping is two steps. Insome embodiments, a step of capping is more than two steps.

In some embodiments, a step of capping comprises steps of capping thefree amine of the chiral auxiliary and capping any residual unreacted 5′hydroxyl groups. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with the samecapping group. In some embodiments, the free amine of the chiralauxiliary and the unreacted 5′ hydroxyl groups are capped with differentcapping groups. In certain embodiments, capping with different cappinggroups allows for selective removal of one capping group over the otherduring synthesis of the oligonucleotide. In some embodiments, thecapping of both groups occurs simultaneously. In some embodiments, thecapping of both groups occurs iteratively.

In certain embodiments, capping occurs iteratively and comprises a firststep of capping the free amine followed by a second step of capping thefree 5′ hydroxyl group, wherein both the free amine and the 5′ hydroxylgroup are capped with the same capping group. For instance, in someembodiments, the free amine of the chiral auxiliary is capped using ananhydride (e.g., phenoxyacetic anhydride, i.e., Pac₂O) prior to cappingof the 5′ hydroxyl group with the same anhydride. In certainembodiments, the capping of the 5′ hydroxyl group with the sameanhydride occurs under different conditions (e.g., in the presence ofone or more additional reagents). In some embodiments, capping of the 5′hydroxyl group occurs in the presence of an amine base in an etherialsolvent (e.g., NMI (N-methylimidazole) in THF). The phrase “cappinggroup” is used interchangeably herein with the phrases “protectinggroup” and “blocking group”.

In some embodiments, an amine capping group is characterized in that iteffectively caps the amine such that it prevents rearrangement and/ordecomposition of the intermediate phosphite species. In someembodiments, a capping group is selected for its ability to protect theamine of the chiral auxiliary in order to prevent intramolecularcleavage of the internucleotide linkage phosphorus.

In some embodiments, a 5′ hydroxyl group capping group is characterizedin that it effectively caps the hydroxyl group such that it prevents theoccurrence of “shortmers,” e.g., “n-m” (m and n are integers and m<n; nis the number of bases in the targeted oligonucleotide) impurities thatoccur from the reaction of an oligonucleotide chain that fails to reactin a first cycle but then reacts in one or more subsequent cycles. Thepresence of such shortmers, especially “n−1”, has a deleterious effectupon the purity of the crude oligonucleotide and makes finalpurification of the oligonucleotide tedious and generally low-yielding.

In some embodiments, a particular cap is selected based on its tendencyto facilitate a particular type of reaction under particular conditions.For instance, in some embodiments, a capping group is selected for itsability to facilitate an E1 elimination reaction, which reaction cleavesthe cap and/or auxiliary from the growing oligonucleotide. In someembodiments, a capping group is selected for its ability to facilitatean E2 elimination reaction, which reaction cleaves the cap and/orauxiliary from the growing oligonucleotide. In some embodiments, acapping group is selected for its ability to facilitate a β-eliminationreaction, which reaction cleaves the cap and/or auxiliary from thegrowing oligonucleotide.

Modifying Step:

As used herein, the phrase “modifying step”, “modification step” and“P-modification step” are used interchangeably and refer generally toany one or more steps used to install a modified internucleotidiclinkage. In some embodiments, the modified internucleotidic linkagehaving the structure of formula I. A P-modification step of the presentdisclosure occurs during assembly of a provided oligonucleotide ratherthan after assembly of a provided oligonucleotide is complete. Thus,each nucleotide unit of a provided oligonucleotide can be individuallymodified at the linkage phosphorus during the cycle within which thenucleotide unit is installed.

In some embodiments, a suitable P-modification reagent is a sulfurelectrophile, selenium electrophile, oxygen electrophile, boronatingreagent, or an azide reagent.

For instance, in some embodiments, a selemium reagent is elementalselenium, a selenium salt, or a substituted diselenide. In someembodiments, an oxygen electrophile is elemental oxygen, peroxide, or asubstituted peroxide. In some embodiments, a boronating reagent is aborane-amine (e.g., N,N-diisopropylethylamine (BH₃-DIPEA),borane-pyridine (BH₃.Py), borane-2-chloropyridine (BH₃.CPy),borane-aniline (BH₃.An)), a borane-ether reagent (e.g.,borane-tetrahydrofuran (BH₃-THF)), a borane-dialkylsulfide reagent(e.g., BH₃-Me₂S), aniline-cyanoborane, or atriphenylphosphine-carboalkoxyborane. In some embodiments, an azidereagent is comprises an azide group capable of undergoing subsequentreduction to provide an amine group.

In some embodiments, a P-modification reagent is a sulfurization reagentas described herein. In some embodiments, a step of modifying comprisessulfurization of phosphorus to provide a phosphorothioate linkage orphosphorothioate triester linkage. In some embodiments, a step ofmodifying provides an oligonucleotide having an internucleotidic linkageof formula I.

In some embodiments, the present disclosure provides sulfurizingreagents, and methods of making, and use of the same.

In some embodiments, such sulfurizing reagents are thiosulfonatereagents. In some embodiments, a thiosulfonate reagent has a structureof formula S-I:

wherein:

R^(s1) is R; and

each of R, L and R¹ is independently as defined and described above andherein.

In some embodiments, the sulfurizing reagent is a bis(thiosulfonate)reagent. In some embodiments, the bis(thiosulfonate) reagent has thestructure of formula S-II:

wherein each of R^(s1) and L is independently as defined and describedabove and herein.

As defined generally above, R^(s1) is R, wherein R is as defined anddescribed above and herein. In some embodiments, R^(s1) is optionallysubstituted aliphatic, aryl, heterocyclyl or heteroaryl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is optionally substituted alkyl. In someembodiments, R^(s1) is methyl. In some embodiments, R^(s1) iscyanomethyl. In some embodiments, R^(s1) is nitromethyl. In someembodiments, R^(s1) is optionally substituted aryl. In some embodiments,R^(s1) is optionally substituted phenyl. In some embodiments, R^(s1) isphenyl. In some embodiments, R^(s1) is p-nitrophenyl. In someembodiments, R^(s1) is p-methylphenyl. In some embodiments, R^(s1) isp-chlorophenyl. In some embodiments, R^(s1) is o-chlorophenyl. In someembodiments, R^(s1) is 2,4,6-trichlorophenyl. In some embodiments,R^(s1) is pentafluorophenyl. In some embodiments, R^(s1) is optionallysubstituted heterocyclyl. In some embodiments, R^(s1) is optionallysubstituted heteroaryl.

In some embodiments, R^(s1)—S(O)₂S— is

In someembodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, R^(s1)—S(O)₂S— is

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is —S—R^(L3)— or —S—C(O)—R^(L3)—. In some embodiments, Lis —S—R^(L3)— or —S—C(O)—R^(L3)—, wherein R^(L3) is an optionallysubstituted C₁-C₆ alkylene. In some embodiments, L is —S—R^(L3)— or—S—C(O)—R^(L3)—, wherein R^(L3) is an optionally substituted C₁-C₆alkenylene. In some embodiments, L is —S—R^(L3)— or —S—C(O)—R^(L3)—,wherein R^(L3) is an optionally substituted C₁-C₆ alkylene wherein oneor more methylene units are optionally and independently replaced by anoptionally substituted C₁-C₆ alkenylene, arylene, or heteroarylene. Insome embodiments, In some embodiments, R^(L3) is an optionallysubstituted —S—(C₁-C₆ alkenylene)-, —S—(C₁-C₆ alkylene)-, —S—(C₁-C₆alkylene)-arylene-(C₁-C₆ alkylene)-, —S—CO-arylene-(C₁-C₆ alkylene)-, or—S—CO—(C₁-C₆ alkylene)-arylene-(C₁-C₆ alkylene)-. In some embodiments,the sulfurizing reagent has the structure of S-I or S-II, wherein L is—S—R^(L3)— or —S—C(O)—R^(L3)—, and the sulfur atom is connected to R¹.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is alkylene, alkenylene, arylene or heteroarylene.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein L is

In some embodiments, L is

wherein the sulfur atom is connected to R¹. In some embodiments, thesulfurizing reagent has the structure of S-I or S-II, wherein R¹ is

In some embodiments, R¹ is

wherein the sulfur atom is connected to L. In some embodiments, thesulfurizing reagent has the structure of S-I or S-II, wherein L is

wherein the sulfur atom is connected to R¹; and R¹ is

wherein the sulfur atom is connected to L.

In some embodiments, the sulfurizing reagent has the structure of S-I orS-II, wherein R¹ is —S—R^(L2), wherein R^(L2) is as defined anddescribed above and herein. In some embodiments, R^(L2) is an optionallysubstituted group selected from —S—(C₁-C₆ alkylene)-heterocyclyl,—S—(C₁-C₆ alkenylene)-heterocyclyl, —S—(C₁-C₆ alkylene)-N(R′)₂,—S—(C₁-C₆ alkylene)-N(R′)₃, wherein each R′ is as defined above anddescribed herein.

In some embodiments, -L-R¹ is —R^(L3)—S—S—R^(L2), wherein each variableis independently as defined above and described herein. In someembodiments, -L-R¹ is —R^(L3)—C(O)—S—S—R^(L2), wherein each variable isindependently as defined above and described herein.

Example bis(thiosulfonate) reagents of formula S—II are depicted below:

In some embodiments, the sulfurization reagent is a compound having oneof the following formulae:

S₈, R^(s2)—S—S—R^(s3), or R^(s2)—S—X^(s)—R^(s3),

wherein:

each of R^(s2) and R^(s3) is independently an optionally substitutedgroup selected from aliphatic, aminoalkyl, carbocyclyl, heterocyclyl,heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy,acyl, amide, imide, or thiocarbonyl; or

R^(s2) and R^(s3) are taken together with the atoms to which they arebound to form an optionally substituted heterocyclic or heteroaryl ring;

X^(s) is —S(O)₂—, —O—, or —N(R′)—; and

R′ is as defined and described above and herein.

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is S₈,

In some embodiments, the sulfurization reagent is

Example sulfuring reagents are depicted in Table 5 below.

TABLE 5 Example sulfurization reagents.

S₈

POS

In some embodiments, a provided sulfurization reagent is used to modifyan H-phosphonate. For instance, in some embodiments, an H-phosphonateoligonucleotide is synthesized using, e.g., a method of Wada I or WadaII, and is modified using a sulfurization reagent of formula S—I orS-II:

wherein each of R^(s1), L, and R¹ are as described and defined above andherein.

In some embodiments, the present disclosure provides a process forsynthesizing a phosphorothioate triester, comprising steps of: i)reacting an H-phosphonate of structure:

wherein each of W, Y, and Z are as described and defined above andherein, with a silylating reagent to provide a silyloxyphosphonate; andii) reacting the silyloxyphosphonate with a sulfurization reagent ofstructure S—I or S-II:

to provide a phosphorothiotriester.

In some embodiments, a selenium electrophile is used instead of asulfurizing reagent to introduce modification to the internucleotidiclinkage. In some embodiments, a selenium electrophile is a compoundhaving one of the following formulae:

Se, R^(s2)—Se—Se—R^(s3), or R^(s2)—Se—X^(s)—R^(s3),

wherein:

each of R^(s2) and R^(s3) is independently an optionally substitutedgroup selected from aliphatic, aminoalkyl, carbocyclyl, heterocyclyl,heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy,acyl, amide, imide, or thiocarbonyl; or

R^(s2) and R^(s3) are taken together with the atoms to which they arebound to form an optionally substituted heterocyclic or heteroaryl ring;

X^(s) is —S(O)₂—, —O—, or —N(R′)—; and

R′ is as defined and described above and herein.

In other embodiments, the selenium electrophile is a compound of Se,KSeCN,

In some embodiments, the selenium electrophile is Se or

In some embodiments, a sulfurization reagent for use in accordance withthe present disclosure is characterized in that the moiety transferredto phosphorus during sulfurization is a substituted sulfur (e.g., —SR)as opposed to a single sulfur atom (e.g., —S— or ═S).

In some embodiments, a sulfurization reagent for use in accordance withthe present disclosure is characterized in that the activity of thereagent is tunable by modifying the reagent with a certain electronwithdrawing or donating group.

In some embodiments, a sulfurization reagent for use in accordance withthe present disclosure is characterized in that it is crystalline. Insome embodiments, a sulfurization reagent for use in accordance with thepresent disclosure is characterized in that it has a high degree ofcrystallinity. In certain embodiments, a sulfurization reagent for usein accordance with the present disclosure is characterized by ease ofpurification of the reagent via, e.g., recrystallization. In certainembodiments, a sulfurization reagent for use in accordance with thepresent disclosure is characterized in that it is substantially freefrom sulfur-containing impurities. In some embodiments, sulfurizationreagents which are substantially free from sulfur-containing impuritiesshow increased efficiency.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages. To synthesize suchchirally controlled oligonucleotides, one or more modifying steps areoptionally replaced with an oxidation step to install the correspondingphosphate diester linkages. In some embodiments, the oxidation step isperformed in a fashion similar to ordinary oligonucleotide synthesis. Insome embodiments, an oxidation step comprises the use of I₂. In someembodiments, an oxidation step comprises the use of I₂ and pyridine. Insome embodiments, an oxidation step comprises the use of 0.02 M 12 in aTHF/pyridine/water (70:20:10—v/v/v) co-solvent system. An example cycleis depicted in Scheme I-c.

In some embodiments, a phosphorothioate is directly formed throughsulfurization by a sulfurization reagents, e.g.,3-phenyl-1,2,4-dithiazolin-5-one. In some embodiments, after a directinstallation of a phosphorothioate, a chiral auxiliary group remainsattached to the internucleotidic phosphorus atom. In some embodiments,an additional de-protecting step is required to remove the chiralauxiliary (e.g., for DPSE-type chiral auxiliary, using TBAF, HF-Et₃N,etc.).

In some embodiments, a phosphorothioate precursor is used to synthesizechirally controlled oligonucleotides comprising phosphorothioatelinkages. In some embodiments, such a phosphorothioate precursor is

In some embodiments,

is converted into phosphorothioate diester linkages during standarddeprotection/release procedure after cycle exit. Examples are furtherdepicted below.

In some embodiments, the provided chirally controlled oligonucleotidecomprises one or more phosphate diester linkages and one or morephosphorothioate diester linkages. In some embodiments, the providedchirally controlled oligonucleotide comprises one or more phosphatediester linkages and one or more phosphorothioate diester linkages,wherein at least one phosphate diester linkage is installed after allthe phosphorothioate diester linkages when synthesized from 3′ to 5′. Tosynthesize such chirally controlled oligonucleotides, in someembodiments, one or more modifying steps are optionally replaced with anoxidation step to install the corresponding phosphate diester linkages,and a phosphorothioate precursor is installed for each of thephosphorothioate diester linkages. In some embodiments, aphosphorothioate precursor is converted to a phosphorothioate diesterlinkage after the desired oligonucleotide length is achieved. In someembodiments, the deprotection/release step during or after cycle exitconverts the phosphorothioate precursors into phosphorothioate diesterlinkages. In some embodiments, a phosphorothioate precursor ischaracterized in that it has the ability to be removed by abeta-elimination pathway. In some embodiments, a phosphorothioateprecursor is

As understood by one of ordinary skill in the art, one of the benefitsof using a phosphorothioate precursor, for instance,

during synthesis is that

is more stable than phosphorothioate in certain conditions.

In some embodiments, a phosphorothioate precursor is a phosphorusprotecting group as described herein, e.g., 2-cyanoethyl (CE or Cne),2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl,o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl,3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl,2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl,2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl,4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl. Examples are furtherdepicted below.

As noted above, in some embodiments, sulfurization occurs underconditions which cleave the chiral reagent from the growingoligonucleotide. In some embodiments, sulfurization occurs underconditions which do not cleave the chiral reagent from the growingoligonucleotide.

In some embodiments, a sulfurization reagent is dissolved in a suitablesolvent and delivered to the column. In certain embodiments, the solventis a polar aprotic solvent such as a nitrile solvent. In someembodiments, the solvent is acetonitrile. In some embodiments, asolution of sulfurization reagent is prepared by mixing a sulfurizationreagent (e.g., a thiosulfonate derivative as described herein) withBSTFA (N,O-bis-trimethylsilyl-trifluoroacetamide) in a nitrile solvent(e.g., acetonitrile). In some embodiments, BSTFA is not included. Forexample, the present inventors have found that relatively more reactivesulfurization reagents of general formula R^(s2)—S—S(O)₂—R^(s3) canoften successfully participate in sulfurization reactions in the absenceof BSTFA. To give but one example, the inventors have demonstrated thatwhere R^(s2) is p-nitrophenyl and R^(s3) is methyl then no BSTFA isrequired. In light of this disclosure, those skilled in the art willreadily be able to determine other situations and/or sulfurizationreagents that do not require BSTFA.

In some embodiments, the sulfurization step is performed at roomtemperature. In some embodiments, the sulfurization step is performed atlower temperatures such as about 0° C., about 5° C., about 10° C., orabout 15° C. In some embodiments, the sulfurization step is performed atelevated temperatures of greater than about 20° C.

In some embodiments, a sulfurization reaction is run for about 1 minuteto about 120 minutes. In some embodiments, a sulfurization reaction isrun for about 1 minute to about 90 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 60 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 30 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 25 minutes. In some embodiments, asulfurization reaction is run for about 1 minute to about 20 minutes. Insome embodiments, a sulfurization reaction is run for about 1 minute toabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 1 minute to about 10 minutes. In some embodiments, asulfurization reaction is run for about 5 minute to about 60 minutes.

In some embodiments, a sulfurization reaction is run for about 5minutes. In some embodiments, a sulfurization reaction is run for about10 minutes. In some embodiments, a sulfurization reaction is run forabout 15 minutes. In some embodiments, a sulfurization reaction is runfor about 20 minutes. In some embodiments, a sulfurization reaction isrun for about 25 minutes. In some embodiments, a sulfurization reactionis run for about 30 minutes. In some embodiments, a sulfurizationreaction is run for about 35 minutes. In some embodiments, asulfurization reaction is run for about 40 minutes. In some embodiments,a sulfurization reaction is run for about 45 minutes. In someembodiments, a sulfurization reaction is run for about 50 minutes. Insome embodiments, a sulfurization reaction is run for about 55 minutes.In some embodiments, a sulfurization reaction is run for about 60minutes.

It was unexpectedly found that certain of the sulfurization modificationproducts made in accordance with methods of the present disclosure areunexpectedly stable. In some embodiments, it the unexpectedly stableproducts are phosphorothioate triesters. In some embodiments, theunexpectedly stable products are chirally controlled oligonucleotidescomprising one or more internucleotidic linkages having the structure offormula I-c.

One of skill in the relevant arts will recognize that sulfurizationmethods described herein and sulfurization reagents described herein arealso useful in the context of modifying H-phosphonate oligonucleotidessuch as those described in Wada II (WO2010/064146).

In some embodiments, the sulfurization reaction has a stepwisesulfurization efficiency that is at least about 80%, 85%, 90%, 95%, 96%,97%, or 98%. In some embodiments, the sulfurization reaction provides acrude dinucleotide product composition that is at least 98% pure. Insome embodiments, the sulfurization reaction provides a crudetetranucleotide product composition that is at least 90% pure. In someembodiments, the sulfurization reaction provides a crudedodecanucleotide product composition that is at least 70% pure. In someembodiments, the sulfurization reaction provides a crude icosanucleotideproduct composition that is at least 50% pure.

Once the step of modifying the linkage phosphorus is complete, theoligonucleotide undergoes another deblock step in preparation forre-entering the cycle. In some embodiments, a chiral auxiliary remainsintact after sulfurization and is deblocked during the subsequentdeblock step, which necessarily occurs prior to re-entering the cycle.The process of deblocking, coupling, capping, and modifying, arerepeated until the growing oligonucleotide reaches a desired length, atwhich point the oligonucleotide can either be immediately cleaved fromthe solid support or left attached to the support for purificationpurposes and later cleaved. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleaveageof the oligonucleotide from the support and deprotection of the basesoccurs in a single step. In some embodiments, one or more protectinggroups are present on one or more of the nucleotide bases, and cleaveageof the oligonucleotide from the support and deprotection of the basesoccurs in more than one step. In some embodiments, deprotection andcleavage from the support occurs under basic conditions using, e.g., oneor more amine bases. In certain embodiments, the one or more amine basescomprise propyl amine. In certain embodiments, the one or more aminebases comprise pyridine.

In some embodiments, cleavage from the support and/or deprotectionoccurs at elevated temperatures of about 30° C. to about 90° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 40° C. to about 80° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 50° C. to about 70° C. In someembodiments, cleavage from the support and/or deprotection occurs atelevated temperatures of about 60° C. In some embodiments, cleavage fromthe support and/or deprotection occurs at ambient temperatures.

Example purification procedures are described herein and/or are knowngenerally in the relevant arts.

Noteworthy is that the removal of the chiral auxiliary from the growingoligonucleotide during each cycle is beneficial for at least the reasonsthat (1) the auxiliary will not have to be removed in a separate step atthe end of the oligonucleotide synthesis when potentially sensitivefunctional groups are installed on phosphorus; and (2) unstablephosphorus-auxiliary intermediates prone to undergoing side reactionsand/or interfering with subsequent chemistry are avoided. Thus, removalof the chiral auxiliary during each cycle makes the overall synthesismore efficient.

While the step of deblocking in the context of the cycle is describedabove, additional general methods are included below.

Deblocking Step

In some embodiments, the step of coupling is preceded by a step ofdeblocking. For instance, in some embodiments, the 5′ hydroxyl group ofthe growing oligonucleotide is blocked (i.e., protected) and must bedeblocked in order to subsequently react with a nucleoside couplingpartner.

In some embodiments, acidification is used to remove a blocking group.In some embodiments, the acid is a Brønsted acid or Lewis acid. UsefulBrønsted acids are carboxylic acids, alkylsulfonic acids, arylsulfonicacids, phosphoric acid and its derivatives, phosphonic acid and itsderivatives, alkylphosphonic acids and their derivatives, arylphosphonicacids and their derivatives, phosphinic acid, dialkylphosphinic acids,and diarylphosphinic acids which have a pKa (25° C. in water) value of−0.6 (trifluoroacetic acid) to 4.76 (acetic acid) in an organic solventor water (in the case of 80% acetic acid). The concentration of the acid(1 to 80%) used in the acidification step depends on the acidity of theacid. Consideration to the acid strength must be taken into account asstrong acid conditions will result in depurination/depyrimidination,wherein purinyl or pyrimidinyl bases are cleaved from ribose ring and orother sugar ring. In some embodiments, an acid is selected fromR^(a1)COOH, R^(a1)SO₃H, R^(a3)SO₃H,

wherein each of R^(a1) and R^(a2) is independently hydrogen or anoptionally substituted alkyl or aryl, and R^(a3) is an optionallysubstituted alkyl or aryl.

In some embodiments, acidification is accomplished by a Lewis acid in anorganic solvent. Example such useful Lewis acids are Zn(X^(a))₂ whereinX^(a) is Cl, Br, I, or CF₃SO₃.

In some embodiments, the step of acidifying comprises adding an amountof a Brønsted or Lewis acid effective to remove a blocking group withoutremoving purine moieties from the condensed intermediate.

Acids that are useful in the acidifying step also include, but are notlimited to 10% phosphoric acid in an organic solvent, 10% hydrochloricacid in an organic solvent, 1% trifluoroacetic acid in an organicsolvent, 3% dichloroacetic acid or trichloroacetic acid in an organicsolvent or 80% acetic acid in water. The concentration of any Brønstedor Lewis acid used in this step is selected such that the concentrationof the acid does not exceed a concentration that causes cleavage of anucleobase from a sugar moiety.

In some embodiments, acidification comprises adding 1% trifluoroaceticacid in an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 8% trifluoroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 3%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding about 0.1% to about 10%dichloroacetic acid or trichloroacetic acid in an organic solvent. Insome embodiments, acidification comprises adding 3% trichloroacetic acidin an organic solvent. In some embodiments, acidification comprisesadding about 0.1% to about 10% trichloroacetic acid in an organicsolvent. In some embodiments, acidification comprises adding 80% aceticacid in water. In some embodiments, acidification comprises adding about50% to about 90%, or about 50% to about 80%, about 50% to about 70%,about 50% to about 60%, about 70% to about 90% acetic acid in water. Insome embodiments, the acidification comprises the further addition ofcation scavengers to an acidic solvent. In certain embodiments, thecation scavengers can be triethylsilane or triisopropylsilane. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 1% trifluoroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% dichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in an organic solvent. In someembodiments, a blocking group is deblocked by acidification, whichcomprises adding 3% trichloroacetic acid in dichloromethane.

In certain embodiments, methods of the present disclosure are completedon a synthesizer and the step of deblocking the hydroxyl group of thegrowing oligonucleotide comprises delivering an amount solvent to thesynthesizer column, which column contains a solid support to which theoligonucleotide is attached. In some embodiments, the solvent is ahalogenated solvent (e.g., dichloromethane). In certain embodiments, thesolvent comprises an amount of an acid. In some embodiments, the solventcomprises an amount of an organic acid such as, for instance,trichloroacetic acid. In certain embodiments, the acid is present in anamount of about 1% to about 20% w/v. In certain embodiments, the acid ispresent in an amount of about 1% to about 10% w/v. In certainembodiments, the acid is present in an amount of about 1% to about 5%w/v. In certain embodiments, the acid is present in an amount of about 1to about 3% w/v. In certain embodiments, the acid is present in anamount of about 3% w/v. Methods for deblocking a hydroxyl group aredescribed further herein. In some embodiments, the acid is present in 3%w/v is dichloromethane.

In some embodiments, the chiral auxiliary is removed before thedeblocking step. In some embodiments, the chiral auxiliary is removedduring the deblocking step.

In some embodiments, cycle exit is performed before the deblocking step.In some embodiments, cycle exit is preformed after the deblocking step.

General Conditions for Blocking Group/Protecting Group Removal

Functional groups such as hydroxyl or amino moieties which are locatedon nucleobases or sugar moieties are routinely blocked with blocking(protecting) groups (moieties) during synthesis and subsequentlydeblocked. In general, a blocking group renders a chemical functionalityof a molecule inert to specific reaction conditions and can later beremoved from such functionality in a molecule without substantiallydamaging the remainder of the molecule (see e.g., Green and Wuts,Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, NewYork, 1991). For example, amino groups can be blocked with nitrogenblocking groups such as phthalimido, 9-fludrenylmethoxycarbonyl (FMOC),triphenylmethylsulfenyl, t-BOC, 4,4′-dimethoxytrityl (DMTr),4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr), or9-(p-methoxyphenyl)xanthin-9-yl (MOX). Carboxyl groups can be protectedas acetyl groups. Hydroxy groups can be protected such astetrahydropyranyl (THP), t-butyldimethylsilyl (TBDMS),1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp),1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp),1-(2-chloroethoxy)ethyl, 3-methoxy-1,5-dicarbomethoxypentan-3-yl (MDP),bis(2-acetoxyethoxy)methyl (ACE), triisopropylsilyloxymethyl (TOM),1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM),[4-(N-dichloroacetyl-N-methylamino)benzyloxy]methyl, 2-cyanoethyl (CN),pivaloyloxymethyl (PivOM), levunyloxymethyl (ALE). Other representativehydroxyl blocking groups have been described (see e.g., Beaucage et al.,Tetrahedron, 1992, 46, 2223). In some embodiments, hydroxyl blockinggroups are acid-labile groups, such as the trityl, monomethoxytrityl,dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and9-(p-methoxyphenyl)xanthin-9-yl (MOX). Chemical functional groups canalso be blocked by including them in a precursor form. Thus an azidogroup can be considered a blocked form of an amine as the azido group iseasily converted to the amine. Further representative protecting groupsutilized in nucleic acid synthesis are known (see e.g. Agrawal et al.,Protocols for Oligonucleotide Conjugates, Eds., Humana Press, NewJersey, 1994, Vol. 26, pp. 1-72).

Various methods are known and used for removal of blocking groups fromnucleic acids. In some embodiments, all blocking groups are removed. Insome embodiments, a portion of blocking groups are removed. In someembodiments, reaction conditions can be adjusted to selectively removecertain blocking groups.

In some embodiments, nucleobase blocking groups, if present, arecleavable with an acidic reagent after the assembly of a providedoligonucleotide. In some embodiment, nucleobase blocking groups, ifpresent, are cleavable under neither acidic nor basic conditions, e.g.cleavable with fluoride salts or hydrofluoric acid complexes. In someembodiments, nucleobase blocking groups, if present, are cleavable inthe presence of base or a basic solvent after the assembly of a providedoligonucleotide. In certain embodiments, one or more of the nucleobaseblocking groups are characterized in that they are cleavable in thepresence of base or a basic solvent after the assembly of a providedoligonucleotide but are stable to the particular conditions of one ormore earlier deprotection steps occurring during the assembly of theprovided oligonucleotide.

In some embodiments, blocking groups for nucleobases are not required.In some embodiments, blocking groups for nucleobases are required. Insome embodiments, certain nucleobases require one or more blockinggroups while other nucleobases do not require one or more blockinggroups.

In some embodiments, the oligonucleotide is cleaved from the solidsupport after synthesis. In some embodiments, cleavage from the solidsupport comprises the use of propylamine. In some embodiments, cleavagefrom the solid support comprises the use of propylamine in pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in pyridine. In some embodiments, cleavage from thesolid support comprises the use of propylamine in anhydrous pyridine. Insome embodiments, cleavage from the solid support comprises the use of20% propylamine in anhydrous pyridine. In some embodiments, cleavagefrom the solid support comprises use of a polar aprotic solvent such asacetonitrile, NMP, DMSO, sulfone, and/or lutidine. In some embodiments,cleavage from the solid support comprises use of solvent, e.g., a polaraprotic solvent, and one or more primary amines (e.g., a C₁₋₁₀ amine),and/or one or more of methoxylamine, hydrazine, and pure anhydrousammonia.

In some embodiments, deprotection of oligonucleotide comprises the useof propylamine. In some embodiments, deprotection of oligonucleotidecomprises the use of propylamine in pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inpyridine. In some embodiments deprotection of oligonucleotide comprisesthe use of propylamine in anhydrous pyridine. In some embodiments,deprotection of oligonucleotide comprises the use of 20% propylamine inanhydrous pyridine.

In some embodiments, the oligonucleotide is deprotected during cleavage.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about room temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at elevated temperature.In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at above about 30° C., 40°C., 50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at about 30° C., 40° C.,50° C., 60° C., 70° C., 80° C. 90° C. or 100° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 40-80° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 50-70° C. In some embodiments,cleavage of oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at about 60° C.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for more than 0.1 hr, 1hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 0.1-5 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 3-10 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5-15 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10-20 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15-25 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 20-40 hrs. Insome embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 2 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 5 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 10 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 15 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 18 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed for about 24 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature formore than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30hrs, or 40 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed at roomtemperature for about 5-48 hrs. In some embodiments, cleavage ofoligonucleotide from solid support, or deprotection of oligonucleotide,is performed at room temperature for about 10-24 hrs. In someembodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide, is performed at room temperature forabout 18 hrs. In some embodiments, cleavage of oligonucleotide fromsolid support, or deprotection of oligonucleotide, is performed atelevated temperature for more than 0.1 hr, 1 hr, 2 hrs, 5 hrs, 10 hrs,15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40 hrs. In some embodiments, cleavageof oligonucleotide from solid support, or deprotection ofoligonucleotide, is performed at elevated temperature for about 0.5-5hrs. In some embodiments, cleavage of oligonucleotide from solidsupport, or deprotection of oligonucleotide, is performed at about 60°C. for about 0.5-5 hrs. In some embodiments, cleavage of oligonucleotidefrom solid support, or deprotection of oligonucleotide, is performed atabout 60° C. for about 2 hrs.

In some embodiments, cleavage of oligonucleotide from solid support, ordeprotection of oligonucleotide comprises the use of propylamine and isperformed at room temperature or elevated temperature for more than 0.1hr, 1 hr, 2 hrs, 5 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs, 30 hrs, or 40hrs. Example conditions are 20% propylamine in pyridine at roomtemperature for about 18 hrs, and 20% propylamine in pyridine at 60° C.for about 18 hrs.

In some embodiments, prior to cleavage from solid support, a step isperformed to remove a chiral auxiliary group, if one is still attachedto an internucleotidic phosphorus atom. In some embodiments, forexample, one or more DPSE type chiral auxiliary groups remain attachedto internucleotidic phosphorus atoms during the oligonucleotidesynthesis cycle. Suitable conditions for removing remaining chiralauxiliary groups are widely known in the art, e.g., those described inWada I, Wada II, Wada III, Chiral Control, etc. In some embodiments, acondition for removing DPSE type chiral auxiliary is TBAF or HF-Et₃N,e.g., 0.1M TBAF in MeCN, 0.5M HF-Et₃N in THF or MeCN, etc. In someembodiments, the present disclosure recognizes that a linker may becleaved during the process of removing a chiral auxiliary group. In someembodiments, the present disclosure provides linkers, such as the SPlinker, that provides better stability during chiral auxiliary groupremoval. Among other things, certain linkers provided by the presentdisclosure provided improved yield and/or purity.

In some embodiments, an activator is a “Wada” activator, i.e., theactivator is from any one of Wada I, II, or III documents cited above.

Example activating groups are depicted below:

In some embodiments an activating reagent is selected from

In some embodiments, an example cycle is depicted in Scheme I-b.

In some embodiments, an example cycle is illustrated in Scheme I-c.

In Scheme I-c, oligonucleotide (or nucleotide, or oligonucleotide withmodified internucleotidic linkage) on solid support (C-1) is coupledwith phosphoramidite C-2. After coupling and capping, an oxidation stepis performed. After deblocking, a phosphate diester linkage is formed.The cycle product C-3 can either re-enter cycle C to install morephosphate diester linkage, or enter other cycles to install other typesof internucleotidic linkages, or go to cycle exit.

In some embodiments, non-chirally pure phosphoramidite can be usedinstead of C-2 in Scheme I-c. In some embodiments,(3-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the phosphoramidite being used has the structure of

In some embodiments, the use of a phosphorothioate diester precursorincreases the stability of oligonucleotide during synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe efficiency of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe yield of chirally controlled oligonucleotide synthesis. In someembodiments, the use of a phosphorothioate diester precursor improvesthe product purity of chirally controlled oligonucleotide synthesis.

In some embodiments, the phosphorothioate diester precursor in theabove-mentioned methods is

In some embodiments,

is converted to a phosphorothioate diester linkage duringdeprotection/release. In some embodiments, an example cycle is depictedin Scheme I-d. More examples are depicted below.

As illustrated in Scheme I-d, both phosphorothioate and phosphatediester linkages can be incorporated into the same chirally controlledoligonucleotide. As understood by a person of ordinary skill in the art,the provided methods do not require that the phosphorothioate diesterand the phosphate diester to be consecutive—other internucleotidiclinkages can form between them using a cycle as described above. InScheme I-d, phosphorothioate diester precursors,

are installed in place of the phosphorothioate diester linkages. In someembodiments, such replacement provided increased synthesis efficiencyduring certain steps, for instance, the oxidation step. In someembodiments, the use of phosphorothioate diester precursors generallyimprove the stability of chirally controlled oligonucleotides duringsynthesis and/or storage. After cycle exit, during deprotection/release,the phosphorothioate diester precursor is converted to phosphorothioatediester linkage. In some embodiments, it is beneficial to usephosphorothioate diester precursor even when no phosphate diesterlinkage is present in the chirally controlled oligonucleotide, or nooxidation step is required during synthesis.

As in Scheme I-c, in some embodiments, non-chirally pure phosphoramiditecan be used for cycles comprising oxidation steps. In some embodiments,3-cyanoethylphosphoramidites protected with DMTr is used. In someembodiments, the phosphoramidite being used has the structure of

In some embodiments, methods of the present disclosure provide chirallycontrolled oligonucleotide compositions that are enriched in aparticular oligonucleotide type.

In some embodiments, at least about 10% of a provided crude compositionis of a particular oligonucleotide type. In some embodiments, at leastabout 20% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 30% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 50% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 60% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 70% of a provided crude composition isof a particular oligonucleotide type. In some embodiments, at leastabout 80% of a provided crude composition is of a particularoligonucleotide type. In some embodiments, at least about 90% of aprovided crude composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided crude composition isof a particular oligonucleotide type.

In some embodiments, at least about 1% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 2%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 3% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 4%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 5% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 10%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 20% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 30%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 40% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 50%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 60% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 70%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 80% of a provided composition is of aparticular oligonucleotide type. In some embodiments, at least about 90%of a provided composition is of a particular oligonucleotide type. Insome embodiments, at least about 95% of a provided composition is of aparticular oligonucleotide type.

In some embodiments, an example cycle is depicted in Scheme I-e, below.

In some embodiments, X is H or a 2′-modification. In some embodiments, Xis H or —OR¹, wherein R¹ is not hydrogen. In some embodiments, X is H or—OR¹, wherein R¹ is optionally substituted C₁-6 alkyl. In someembodiments, X is H. In some embodiments, X is —OMe. In someembodiments, X is —OCH₂CH₂OCH₃. In some embodiments, X is —F.

In some embodiments, an example cycle is depicted in Scheme I-f.

In some embodiments, X is H or a 2′-modification. In some embodiments, Xis H or —OR¹, wherein R¹ is not hydrogen. In some embodiments, X is H or—OR¹, wherein R¹ is optionally substituted C₁-6 alkyl. In someembodiments, X is H. In some embodiments, X is —OMe. In someembodiments, X is —OCH₂CH₂OCH₃. In some embodiments, X is —F.

It is understood by a person having ordinary skill in the art thatdifferent types of cycles may be combined to provide complete control ofthe chemical modifications and stereochemistry of oligonucleotides. Insome embodiments, for example, an oligonucleotide synthesis process maycontain one or more Cycles A-F. In some embodiments, a provided methodcomprises at least one cycle using a DPSE-type chiral auxiliary.

In some embodiments, a provided method further comprises providing afluoro-containing reagent. In some embodiments, a providedfluoro-containing reagent removes a chiral reagent, or a product formedfrom a chiral reagent, from oligonucleotides after synthesis. Variousknown fluoro-containing reagents, including those F⁻ sources forremoving —SiR₃ groups, can be utilized in accordance with the presentdisclosure, for example, TBAF, HF₃-Et₃N etc. In some embodiments, afluoro-containing reagent provides better results, for example, shortertreatment time, lower temperature, less de-sulfurization, etc, comparedto traditional methods, such as concentrated ammonia. In someembodiments, for certain fluoro-containing reagent, the presentdisclosure provides linkers for improved results, for example, lesscleavage of oligonucleotides from support during removal of chiralreagent (or product formed therefrom during oligonucleotide synthesis).In some embodiments, a provided linker is an SP linker. In someembodiments, the present disclosure demonstrated that a HF-base complexcan be utilized, such as HF—NR₃, to control cleavage during removal ofchiral reagent (or product formed therefrom during oligonucleotidesynthesis). In some embodiments, HF—NR₃ is HF-NEt₃. In some embodiments,HF—NR₃ enables use of traditional linkers, e.g., succinyl linker.

In some embodiments, the present disclosure comprises a method formanufacturing an oligonucleotide composition directed to a selectedtarget sequence, the method comprising manufacturing a providedoligonucleotide composition comprising a first plurality ofoligonucleotides, each of which has a base sequence complementary to thetarget sequence. In some embodiments, a provided method furthercomprises providing a pharmaceutically acceptable carrier.

Biological Applications and Example Use

In some embodiments, the present disclosure recognizes that properties,e.g., activities, toxicities, etc. of oligonucleotides and compositionsthereof can be optimized by chemical modifications and/orstereochemistry. In some embodiments, the present disclosure providesmethods for optimizing oligonucleotide properties through chemicalmodifications and stereochemistry. In some embodiments, the presentdisclosure provides oligonucleotides and compositions and methodsthereof with low toxicities. In some embodiments, the present disclosureprovides oligonucleotides and compositions and methods thereof with lowtoxicities and enhanced activities (e.g., target-inhibition efficiency,specificity, cleavage rates, cleavage pattern, etc.). In someembodiments, the present disclosure provides oligonucleotides andcompositions and methods thereof with improved protein binding profile.In some embodiments, the present disclosure provides oligonucleotidesand compositions and methods thereof with improved protein bindingprofile and enhanced activities. In some embodiments, the presentdisclosure provides oligonucleotides and compositions and methodsthereof with improved delivery and enhanced activities.

In some embodiments, provided oligonucleotides, compositions and methodshave low toxicities, e.g., when compared to a reference composition. Aswidely known in the art, oligonucleotides can induce toxicities whenadministered to, e.g., cells, tissues, organism, etc. In someembodiments, oligonucleotides can induce undesired immune response. Insome embodiments, oligonucleotide can induce complement activation. Insome embodiments, oligonucleotides can induce activation of thealternative pathway of complement. In some embodiments, oligonucleotidescan induce inflammation. Among other things, the complement system hasstrong cytolytic activity that can damages cells and should therefore bemodulated to reduce potential injuries. In some embodiments,oligonucleotide-induced vascular injury is a recurrent challenge in thedevelopment of oligonucleotides for e.g., pharmaceutical use. In someembodiments, a primary source of inflammation when high doses ofoligonucleotides are administered involves activation of the alternativecomplement cascade. In some embodiments, complement activation is acommon challenge associated with phosphorothioate-containingoligonucleotides, and there is also a potential of some sequences ofphosphorothioates to induce innate immune cell activation. In someembodiments, cytokine release is associated with administration ofoligonucleotides. For example, in some embodiments, increases ininterleukin-6 (IL-6) monocyte chemoattractant protein (MCP-1) and/orinterleukin-12 (IL-12) is observed. See, e.g., Frazier, AntisenseOligonucleotide Therapies: The Promise and the Challenges from aToxicologic Pathologist's Perspective. Toxicol Pathol., 43: 78-89, 2015;and Engelhardt, et al., Scientific and Regulatory Policy CommitteePoints-to-consider Paper: Drug-induced Vascular Injury Associated withNonsmall Molecule Therapeutics in Preclinical Development: Part 2.Antisense Oligonucleotides. Toxicol Pathol. 43: 935-944, 2015.

By controlling of chemical modifications and/or stereochemistry, thepresent disclosure provides improved oligonucleotide compositions andmethods. In some embodiments, provided oligonucleotides comprisechemical modifications. In some embodiments, provided oligonucleotidescomprise base modifications, sugar modifications, internucleotidiclinkage modifications, or any combinations thereof. In some embodiments,provided oligonucleotides comprise base modifications. In someembodiments, provided oligonucleotides comprise sugar modifications. Insome embodiments, provided oligonucleotides comprises 2′-modificationson the sugar moieties. In some embodiments, the present disclosuredemonstrates that 2′-modifications can lower toxicity. In someembodiments, provided oligonucleotides comprises one or more modifiedinternucleotidic linkages and one or more natural phosphate linkages. Insome embodiments, the present disclosure demonstrates that incorporationof one or more natural phosphate linkages into oligonucleotidescomprising one or more modified internucleotidic linkages can lowertoxicity. A natural phosphate linkage can be incorporated into variouslocations of an oligonucleotide. In some embodiments, a naturalphosphate linkage is incorporated into a wing region, or a region closeto the 5′- or the 3′-end. In some embodiments, a natural phosphatelinkage is incorporated into the middle of an oligonucleotide. In someembodiments, a natural phosphate linkage is incorporated into a coreregion. In some embodiments, the present disclosure demonstrates thatstereochemistry, either alone or in combination with chemicalmodifications, can modulate toxicity. In some embodiments, the presentdisclosure demonstrates that stereochemistry, either alone or incombination with chemical modifications, can modulate immune response.In some embodiments, the present disclosure demonstrates thatstereochemistry, either alone or in combination with chemicalmodifications, can modulate complement activation. It is surprisinglyfound that a chirally controlled oligonucleotide composition of anindividual stereoisomer can have dramatically different toxicityprofile, e.g., complement activation, compared to the correspondingstereorandom composition, and/or a chirally controlled oligonucleotidecomposition of another individual stereoisomer. For examples, see FIGS.1-5. In some embodiments, the present disclosure demonstrates thatstereochemistry, either alone or in combination with chemicalmodifications, can modulate complement activation via the alternativepathway. Example chemical modifications, stereochemistry and patternsthereof are extensively described in this disclosure, and they can beused in combinations. Example compositions and methods of are alsoextensively described in this disclosure. A person having ordinary skillin the art understands that methods and compositions described hereincan be used to either increase or decrease immune responses, includingcomplement activation, relative to a reference composition.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering a provided oligonucleotide composition comprising thefirst plurality of oligonucleotides that is characterized by reducedtoxicity relative to a reference oligonucleotide composition of the samecommon nucleotide sequence.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is chirally controlled and that ischaracterized by reduced toxicity relative to a referenceoligonucleotide composition of the same common nucleotide sequence.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition in which eacholigonucleotide in the plurality comprises one or more modified sugarmoieties and the composition is characterized by reduced toxicityrelative to a reference oligonucleotide composition of the same commonnucleotide sequence but lacking at least one of the one or more modifiedsugar moieties.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition in which eacholigonucleotide in the plurality includes one or more natural phosphatelinkages and one or more modified phosphate linkages;

wherein the oligonucleotide composition is characterized by reducedtoxicity when tested in at least one assay that is observed with anotherwise comparable reference composition whose oligonucleotidescomprise fewer natural phosphate linkages.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition in which eacholigonucleotide in the plurality includes one or more natural phosphatelinkages and one or more modified phosphate linkages;

wherein the oligonucleotide composition is characterized by reducedtoxicity when tested in at least one assay that is observed with anotherwise comparable reference composition whose oligonucleotides do notcomprise natural phosphate linkages.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition in which eacholigonucleotide in the plurality comprises one or more modified sugarmoieties and the composition is characterized by reduced toxicityrelative to a reference oligonucleotide composition of the same commonnucleotide sequence but lacking at least one of the one or more modifiedsugar moieties.

In some embodiments, the present disclosure provides a method comprisingsteps of administering to a subject an oligonucleotide compositioncomprising a first plurality of oligonucleotides each of which has acommon base sequence and comprises a modified sugar moiety, wherein theoligonucleotide composition is characterized by reduced toxicity whentested in at least one assay that is observed with an otherwisecomparable reference composition that comprises a reference plurality ofoligonucleotides which have the same common base sequence but have nomodified sugar moieties.

In some embodiments, the present disclosure provides a method comprisingsteps of administering to a subject an oligonucleotide compositioncomprising a first plurality of oligonucleotides each of which has acommon base sequence and comprises one or more natural phosphatelinkages and one or more modified phosphate linkages, wherein theoligonucleotide composition is characterized by reduced toxicity whentested in at least one assay that is observed with an otherwisecomparable reference composition that comprises a reference plurality ofoligonucleotides which have the same common base sequence but have nonatural phosphate linkages.

In some embodiments, the present disclosure provides a method comprisingsteps of administering a chirally controlled oligonucleotide compositionto a subject, wherein the chirally controlled oligonucleotidecomposition is characterized by reduced toxicity when tested in at leastone assay that is observed with an otherwise comparable referencecomposition that includes a different chirally controlledoligonucleotide composition, or a stereorandom oligonucleotidecomposition, comprising oligonucleotides having the same base sequence.

In some embodiments, reduced toxicity is or comprises reduced complementactivation. In some embodiments, reduced toxicity comprises reducedcomplement activation. In some embodiments, reduced toxicity is orcomprises reduced complement activation. In some embodiments, reducedtoxicity comprises reduced complement activation via the alternativepathway. In some embodiments, toxicity can be assessed through measuringlevels of complement activation. In some embodiments, altered complementactivation is observed in an assay that detects a protein whose levelchanges upon complement activation. In some embodiments, alteredcomplement activation is observed in an assay that detects presence,absolute level and or relative levels of one or more complete-activationrelated product. In some embodiments, complement activation is observedin a serum. In some embodiments, complement activation is observed inhuman serum. In some embodiments, complement activation is observed in aprimate serum. In some embodiments, complement activation is observed inmonkey serum.

In some embodiments, complement activation is measured no more than 60minutes after administration of oligonucleotides. In some embodiments,complement activation is measured no more than 50 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured no more than 40 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measuredno more than 30 minutes after administration of oligonucleotides. Insome embodiments, complement activation is measured no more than 25minutes after administration of oligonucleotides. In some embodiments,complement activation is measured no more than 20 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured no more than 15 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measuredno more than 10 minutes after administration of oligonucleotides. Insome embodiments, complement activation is measured no more than 9minutes after administration of oligonucleotides. In some embodiments,complement activation is measured no more than 8 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured no more than 7 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measuredno more than 6 minutes after administration of oligonucleotides. In someembodiments, complement activation is measured no more than 5 minutesafter administration of oligonucleotides. In some embodiments,complement activation is measured no more than 4 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured no more than 3 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measuredno more than 2 minutes after administration of oligonucleotides. In someembodiments, complement activation is measured no more than 1 minuteafter administration of oligonucleotides. In some embodiments,complement activation is measured no more than 30 seconds afteradministration of oligonucleotides. In some embodiments, complementactivation is measured no more than 20 seconds after administration ofoligonucleotides. In some embodiments, complement activation is measuredno more than 10 seconds after administration of oligonucleotides. Insome embodiments, complement activation is measured no more than 5seconds after administration of oligonucleotides.

In some embodiments, complement activation is measured 5 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured 10 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measured15 minutes after administration of oligonucleotides. In someembodiments, complement activation is measured 20 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured 25 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measured30 minutes after administration of oligonucleotides. In someembodiments, complement activation is measured 35 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured 40 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measured45 minutes after administration of oligonucleotides. In someembodiments, complement activation is measured 50 minutes afteradministration of oligonucleotides. In some embodiments, complementactivation is measured 55 minutes after administration ofoligonucleotides. In some embodiments, complement activation is measured60 minutes after administration of oligonucleotides. In someembodiments, complement activation is measured at multiple time pointsafter administration of oligonucleotides, for example, at two or moretime points selected from 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 110 and 120 minutes after administration ofoligonucleotides.

In some embodiments, complement activation is reduced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%. Insome embodiments, complement activation is reduced by at least 5%. Insome embodiments, complement activation is reduced by at least 10%. Insome embodiments, complement activation is reduced by at least 15%. Insome embodiments, complement activation is reduced by at least 20%. Insome embodiments, complement activation is reduced by at least 25%. Insome embodiments, complement activation is reduced by at least 30%. Insome embodiments, complement activation is reduced by at least 35%. Insome embodiments, complement activation is reduced by at least 40%. Insome embodiments, complement activation is reduced by at least 45%. Insome embodiments, complement activation is reduced by at least 50%. Insome embodiments, complement activation is reduced by at least 55%. Insome embodiments, complement activation is reduced by at least 60%. Insome embodiments, complement activation is reduced by at least 65%. Insome embodiments, complement activation is reduced by at least 70%. Insome embodiments, complement activation is reduced by at least 75%. Insome embodiments, complement activation is reduced by at least 80%. Insome embodiments, complement activation is reduced by at least 85%. Insome embodiments, complement activation is reduced by at least 90%. Insome embodiments, complement activation is reduced by at least 95%. Insome embodiments, complement activation is reduced by at least 96%. Insome embodiments, complement activation is reduced by at least 97%. Insome embodiments, complement activation is reduced by at least 98%. Insome embodiments, complement activation is reduced by at least 99%. Insome embodiments, complement activation is reduced to the same level ofa negative control, e.g., water, a buffer not inducing complementactivation, etc. In some embodiments, provided oligonucleotides,compositions and methods reduce injection site inflammation. In someembodiments, provided oligonucleotides, compositions and methods reduceinduced vascular injury.

Markers that can be used to evaluate toxicity are widely known in theart, for example, CH50 (total complement), complement split products(e.g., Bb, C3a, C5a, etc.), MCP-1 and CRP, fibrinogen, haptoglobin,globulin, proteinuria, albuminuria, Angiopoietin-2, Endothelin-1,Eselectin, Thrombospondin-1, Vascular endothelial growth factor alpha,Calponin-1, Tissue inhibitor of metalloproteinase 1, Lipocalin 2,Cytokine-induced neutrophil chemoattractant 1, Alpha-1 acid glycoprotein1, total nitric oxide, Von Willebrands factor, intercellular adhesionmolecule (ICAM), vascular cellular adhesion molecule-1 (VCAM-1),interleukins, monocyte chemotactic protein-1, serum amyloid A, CRP,fibrinogen, plasminogen activator inhibitor-1, caveolin, matrixmetalloproteinases (MMP-1, MMP-3, and MMP-9), vascular endothelialgrowth factor, thrombomodulin, E-selectin, P-selectin, complementpathway analysis and other inflammatory markers (i.e., CRP, MCP-1,MMP-3, and/or other cytokines or chemokines), markers of endothelialactivation (e.g., VCAM), etc. For examples, see Frazier, AntisenseOligonucleotide Therapies: The Promise and the Challenges from aToxicologic Pathologist's Perspective. Toxicol Pathol., 43: 78-89, 2015;Engelhardt, et al., Scientific and Regulatory Policy CommitteePoints-to-consider Paper: Drug-induced Vascular Injury Associated withNonsmall Molecule Therapeutics in Preclinical Development: Part 2.Antisense Oligonucleotides. Toxicol Pathol. 43: 935-944, 2015; etc. Insome embodiments, a marker is protein in the complement pathway. In someembodiments, a marker is protein in the alternative complement pathway.In some embodiments, a marker is protein produced during completeactivation. In some embodiments, a marker is protein produced duringcomplete activation via the alternative pathway. In some embodiments, amarker is selected from C3a, Bb, C4a, C5a, C5b, C6, C7, C8 and C9. Insome embodiments, a marker is selected from C4a, C5a, C5b, C6, C7, C8and C9. In some embodiments, a marker is selected from C3a, C4a, C5a andBb. In some embodiments, a marker is C3a or Bb. In some embodiments, amarker is C3a. In some embodiments, a marker is Bb.

Example assays are widely known in the art, including but not limited tothose described in this disclosure and US2002/0082227; Frazier,Antisense Oligonucleotide Therapies: The Promise and the Challenges froma Toxicologic Pathologist's Perspective. Toxicol Pathol., 43: 78-89,2015; Engelhardt, et al., Scientific and Regulatory Policy CommitteePoints-to-consider Paper: Drug-induced Vascular Injury Associated withNonsmall Molecule Therapeutics in Preclinical Development: Part 2.Antisense Oligonucleotides. Toxicol Pathol. 43: 935-944, 2015; etc.

The present disclosure demonstrates that chirally controlledoligonucleotide compositions of individual stereoisomers can havedifferent complement activation profiles. In some embodiments, chirallycontrolled oligonucleotide compositions of oligonucleotides having allphosphorothioate linkages and a single Rp in the middle may demonstraterelatively high complement activation. As provided in this disclosure,various methods can be used to decrease the relatively high complementactivation of these oligonucleotides, including but not limited tointroduction of one or more natural phosphate linkages. For examples,see FIGS. 4 and 5.

With their improved properties, e.g., low toxicity, high activities,etc., provided oligonucleotides and compositions thereof areparticularly useful for treating various diseases. In some embodiments,provided oligonucleotides, compositions and/or methods are particularlyuseful for reducing a target involved in the complement system. In someembodiments, provided oligonucleotides, compositions and/or methods areparticularly useful for reducing complement activation by reducinglevels of a target in the complement system as providedoligonucleotides, compositions and/or methods themselves only inducelow, if any, complement activation compared to referenceoligonucleotides, compositions and/or methods thereof. In someembodiments, a target involved in the complement system is C1, C1a, C1r,C1s, C1q, MASP-1, MASP-2, C3, C3-convertase, C3a, C3b, C3aR, C4b, C5,C5a, C5aR, Factor B, Factor D, Thrombin, Plasmin, Kallikrein, orFactorXIIa. In some embodiments, provided oligonucleotides, compositionsand/or methods can provide improved treatment of associated diseasessuch as neuroinflammation and neurodegeneration, muscular inflammation,demyelination, vasculitis and nephritis. In some embodiments, a diseaseis a rare disease associated with complement; for examples, see Reis etal., Applying complement therapeutics to rare diseases, ClinicalImmunology (2015), doi: 10.1016/j.clim.2015.08.009. In some embodiments,the present disclosure provides compositions of oligonucleotidestargeting C5. In some embodiments, a provided composition is an siRNAcomposition targeting C5. In some embodiments, the present disclosureprovides compositions of oligonucleotides targeting factor B. In someembodiments, the present disclosure provides compositions and methodstargeting factor B for treatment of lupus nephritis. Example basesequences for targeting factor B include but are not limited to thosedescribed in Grossman et al. Inhibition of the alternative complementpathway by antisense oligonucleotides targeting complement factor Bimproves lupus nephritis in mice. Immunobiology. 2015 Aug. 10. pii:S0171-2985(15)30041-3. doi: 10.1016/j.imbio.2015.08.001.

In some embodiments, the present disclosure provides methods formodulating protein binding properties of oligonucleotides, for example,by adjusting chemical modifications and/or stereochemistry ofoligonucleotides. Example chemical modifications, stereochemistry andcombinations thereof are extensively described in this disclosure. Insome embodiments, the present disclosure provides oligonucleotides andcompositions thereof with improved protein binding profile.

In some embodiments, the present disclosure provides a method,comprising administering a composition comprising a first plurality ofoligonucleotides, which composition displays altered protein binding ascompared with a reference composition comprising a plurality ofoligonucleotides, each of which also has the common base sequence butwhich differs structurally from the oligonucleotides of the firstplurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises:

administering an oligonucleotide composition comprising a firstplurality of oligonucleotides that is characterized by altered proteinbinding relative to a reference oligonucleotide composition of the samecommon nucleotide sequence.

In some embodiments, provided oligonucleotides and compositions increasebeneficial protein binding. In some embodiments, providedoligonucleotides and compositions decrease harmful protein binding. Insome embodiments, provided oligonucleotides and compositions increasebeneficial protein binding and decrease detrimental protein binding.

In some embodiments, improved protein binding profile lower toxicitiesand improve activities of provided oligonucleotides and compositions. Insome embodiments, lowered binding to negative regulators of complementpathways contribute to decreased complement activation. In someembodiments, lowered binding to certain proteins are associated withlower toxicities and/or better activities. In some embodiments,increased binding to certain proteins contributes to lower toxicitiesand/or better activities.

Binding to various proteins, e.g., serum proteins, heparinsulfate-binding proteins, intracellular proteins, etc., byoligonucleotides can be modulated using oligonucleotides, compositionsand methods provided in the present disclosure. Example proteins includebut are not limited to Albumin, Complement Factor H, Factor IX, ApoE,Thrombin, Factor VIIIa, Heparin Cofactor II, alpha-2 macroglobulin,Fibroblast Growth Factor 1, Fibroblast Growth Factor 2, HepatocyteGrowth Factor/Scatter Factor, Vascular Endothelial Growth Factor,High-Mobility Group Protein B1, Cyclophilin B, IL-8 (CXCL8), PlateletFactor 4 (CXCL4), Stromal Cell-Derived Factor-1 (CXCL12), MonocyteChemoattractant Protein-1 (CCL2), Fibroblast Growth Factor Receptor 1,Neuropilin-1, Receptor for Advanced Glycation End Products, ReceptorProtein Tyrosine Phosphatase Sigma, Slit-2, ROBO1, Thrombin,Antithrombin, Protein C inhibitor, Amyloid precursor protein 1,Thrombospondin-1, Annexin A2, PDGF BB, PC4/Sub1, RNF163/ZNF9, Ku70,Ku80, TCP1-alpha, TCP1-beta, TCP1-epsilon, TCP1-gamma, TCP1-Theta,TCP1-delta, HSP90-AA1, HSP90-AB, HSP70-5/GRP78, HSPA1L, HSC70, ACTB,TBBB2C, Vimentin, CArG Binding Factor, DHX30, EIF2S2, EIF4H, GRSF1,hnRNP D1L, hnRNPA1, hnRNPA2, hnRNPH1, hnRNPK, hnRNPQ, hnRNPU, hnRNPUL,ILF2, ILF3, KHSRP, La/SSB, NCL, NPM1, P54nrb, PSF, PSPC1, RHA, YBX1,ACLY, VARS, ANXA2, NDKA, Thymidylate Kinase, JKBP1 delta 6, SHMT2,LRPPRC, NARS, ATAD3A, KCTD12, CD4, GP120, aMb2 (Mac-1), VDAC-1, Ago2 PAZdomain, RAGE, AIM2, DHX36, DHX9, DDX41, IFI16, RIG-I, MDA5, LRRFIP1,DLM-1/ZBP1, TREX1, Laminin, and Fibronectin. In some embodiments, aplurality of oligonucleotides or an oligonucleotide composition cansimultaneously modulate binding to multiple proteins, includingmaintaining the same binding levels to one group of proteins, decreasingbinding levels to another group of proteins, and/or increasing bindinglevel to yet another group of proteins. In some embodiments, providedoligonucleotides, compositions and methods provide increased binding toone or more serum proteins. In some embodiments, a serum protein isalbumin. In some embodiments, provided oligonucleotides, compositionsand methods provide decreased binding to one or more heparinsulfate-binding protein. In some embodiments, provided oligonucleotides,compositions and methods provide decreased binding to one or more FactorH.

In some embodiments, protein binding is decreased by more than 10%. Insome embodiments, protein binding is decreased by more than 20%. In someembodiments, protein binding is decreased by more than 30%. In someembodiments, protein binding is decreased by more than 40%. In someembodiments, protein binding is decreased by more than 50%. In someembodiments, protein binding is decreased by more than 60%. In someembodiments, protein binding is decreased by more than 70%. In someembodiments, protein binding is decreased by more than 75%. In someembodiments, protein binding is decreased by more than 80%. In someembodiments, protein binding is decreased by more than 85%. In someembodiments, protein binding is decreased by more than 90%. In someembodiments, protein binding is decreased by more than 91%. In someembodiments, protein binding is decreased by more than 92%. In someembodiments, protein binding is decreased by more than 93%. In someembodiments, protein binding is decreased by more than 94%. In someembodiments, protein binding is decreased by more than 95%. In someembodiments, protein binding is decreased by more than 96%. In someembodiments, protein binding is decreased by more than 97%. In someembodiments, protein binding is decreased by more than 98%. In someembodiments, protein binding is decreased by more than 99%.

In some embodiments, protein binding is increased by more than 10%. Insome embodiments, protein binding is increased by more than 20%. In someembodiments, protein binding is increased by more than 30%. In someembodiments, protein binding is increased by more than 40%. In someembodiments, protein binding is increased by more than 50%. In someembodiments, protein binding is increased by more than 60%. In someembodiments, protein binding is increased by more than 70%. In someembodiments, protein binding is increased by more than 80%. In someembodiments, protein binding is increased by more than 90%. In someembodiments, protein binding is increased by more than 100%. In someembodiments, protein binding is increased by more than 150%. In someembodiments, protein binding is increased by more than 2 folds. In someembodiments, protein binding is increased by more than 3 folds. In someembodiments, protein binding is increased by more than 4 folds. In someembodiments, protein binding is increased by more than 5 folds. In someembodiments, protein binding is increased by more than 6 folds. In someembodiments, protein binding is increased by more than 7 folds. In someembodiments, protein binding is increased by more than 8 folds. In someembodiments, protein binding is increased by more than 9 folds. In someembodiments, protein binding is increased by more than 10 folds. In someembodiments, protein binding is increased by more than 15 folds. In someembodiments, protein binding is increased by more than 20 folds. In someembodiments, protein binding is increased by more than 25 folds. In someembodiments, protein binding is increased by more than 30 folds. In someembodiments, protein binding is increased by more than 35 folds. In someembodiments, protein binding is increased by more than 40 folds. In someembodiments, protein binding is increased by more than 45 folds. In someembodiments, protein binding is increased by more than 50 folds. In someembodiments, protein binding is increased by more than 60 folds. In someembodiments, protein binding is increased by more than 70 folds. In someembodiments, protein binding is increased by more than 80 folds. In someembodiments, protein binding is increased by more than 90 folds. In someembodiments, protein binding is increased by more than 100 folds.

In some embodiments, the present disclosure provides assays forassessing protein binding. In some embodiments, protein binding can beassessed by binding to albumin. In some embodiments, protein binding canbe assessed by binding to BSA. In some embodiments, protein binding canbe assessed in vitro. Additional suitable assays are widely known in theart.

Chemical modifications, stereochemistry and combinations thereof thatcan improve protein binding profiles are extensively described in thisdisclosure. In some embodiments, more modified internucleotidic linkagescan increase protein binding. In some embodiments, more phosphorothioatelinkages can increase protein binding. In some embodiments, fewermodified internucleotidic linkages can decrease protein binding. In someembodiments, fewer phosphorothioate linkages can decrease proteinbinding. In some embodiments, more Sp chiral internucleotidic linkagesincrease protein binding. In some embodiments, fewer Sp chiralinternucleotidic linkages decrease protein binding. In some embodiments,more modified bases increase protein binding. In some embodiments, fewermodified bases decrease protein binding. In some embodiments, one typeof sugar modifications can increase protein binding compared to theother. In some embodiments, increased 2′-MOE content decrease proteinbiding when compared to 2′-OMe. The present disclosure provides numerouscombinations of chemical modifications and/or stereochemistry patternsto improve protein binding profiles. In some embodiments, the presentdisclosure provides numerous combinations of chemical modificationsand/or stereochemistry patterns to improve protein binding profileswhile at the same time providing lower toxicities and/or betteractivities. Example oligonucleotides and compositions having thesemodifications, stereochemistry, or combinations thereof are describedherein in this disclosure.

Delivery of oligonucleotides to targets can benefit from improvedprotein binding profile. In some embodiments, improved bindingproperties to certain proteins facilitate transportation ofoligonucleotides to target cells, tissues, organs or organism. In someembodiments, improved binding properties to certain proteins promoterelease of oligonucleotides from proteins and other molecules so thatthey can perform their biological functions, including hybridization totarget nucleic acid sequences, inhibition of functions of target nucleicacid sequences, cleavage of target nucleic acid sequences, etc. In someembodiments, provided oligonucleotides, compositions and methods provideimproved uptake of oligonucleotides. In some embodiments, providedoligonucleotides, compositions and methods provide improved uptake ofoligonucleotides.

In some embodiments, the present disclosure provides a method comprisingadministering a composition comprising a first plurality ofoligonucleotides, which composition displays improved delivery ascompared with a reference composition comprising a plurality ofoligonucleotides, each of which also has the common base sequence butwhich differs structurally from the oligonucleotides of the firstplurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

In some embodiments, the present disclosure provides a method ofadministering an oligonucleotide composition comprising a firstplurality of oligonucleotides having a common nucleotide sequence, theimprovement that comprises: administering an oligonucleotide comprisinga first plurality of oligonucleotides that is characterized by improveddelivery relative to a reference oligonucleotide composition of the samecommon nucleotide sequence.

In some embodiments, provided oligonucleotides, compositions and methodsprovide improved systemic delivery. In some embodiments, providedoligonucleotides, compositions and methods provide improvedcytoplasmatic delivery. In some embodiments, improved delivery is to apopulation of cells. In some embodiments, improved delivery is to atissue. In some embodiments, improved delivery is to an organ. In someembodiments, improved delivery is to an organism. Example structuralelements (e.g., chemical modifications, stereochemistry, combinationsthereof, etc.), oligonucleotides, compositions and methods that provideimproved delivery are extensively described in this disclosure.

In some embodiments, the present disclosure provides methods fordecreasing level of a target nucleic acid in a cell, tissue, and/ororganism without low toxicity by contacting with a provided compositionof this disclosure. In some embodiments, the present disclosure providesmethods for decreasing level of a target nucleic acid in a cell, tissue,and/or organism without lower toxicity comparing to a referencecomposition by contacting with a provided composition of thisdisclosure. In some embodiments, the present disclosure provides methodsfor decreasing level of a target nucleic acid in a cell, tissue, and/ororganism without significant complement activation by contacting with aprovided composition of this disclosure. In some embodiments, thepresent disclosure provides methods for decreasing level of a targetnucleic acid in a cell, tissue, and/or organism lower complementactivation compared to a reference composition by contacting with aprovided composition of this disclosure.

In some embodiments, the present disclosure provides methods foridentifying and/or characterizing an oligonucleotide composition withimproved properties, e.g., toxicities, activities, etc. In someembodiments, the present disclosure provides methods for identifyingand/or characterizing an oligonucleotide composition with lowertoxicities compared to a reference composition. In some embodiments, thepresent disclosure provides methods for identifying and/orcharacterizing an oligonucleotide composition with improved proteinbinding profiles compared to a reference composition.

In some embodiments, the present disclosure provides a method ofidentifying and/or characterizing an oligonucleotide composition, themethod comprising steps of:

providing at least one composition comprising a first plurality ofoligonucleotides; and

assessing toxicity relative to a reference composition.

In some embodiments, the present disclosure provides a method ofidentifying and/or characterizing an oligonucleotide composition, themethod comprising steps of:

providing at least one composition comprising a first plurality ofoligonucleotides; and

assessing complement activation relative to a reference composition.

In some embodiments, the present disclosure provides a method ofidentifying and/or characterizing an oligonucleotide composition, themethod comprising steps of:

providing at least one composition comprising a first plurality ofoligonucleotides; and

assessing protein binding profile relative to a reference composition.

In some embodiments, the present disclosure provides a method ofidentifying and/or characterizing an oligonucleotide composition, themethod comprising steps of:

providing at least one composition comprising a first plurality ofoligonucleotides; and

assessing delivery relative to a reference composition.

In some embodiments, the present disclosure provides a method ofidentifying and/or characterizing an oligonucleotide composition, themethod comprising steps of:

providing at least one composition comprising a first plurality ofoligonucleotides; and

assessing cellular uptake relative to a reference composition.

In some embodiments, properties of a provided oligonucleotidecompositions are compared to a reference oligonucleotide composition. Insome embodiments, a reference oligonucleotide composition comprises areference plurality of oligonucleotides.

In some embodiments, a reference oligonucleotide composition is astereorandom oligonucleotide composition. In some embodiments, areference oligonucleotide composition is a stereorandom composition ofoligonucleotides of which all internucleotidic linkages arephosphorothioate. In some embodiments, a reference oligonucleotidecomposition is a DNA oligonucleotide composition with all phosphatelinkages.

In some embodiments, a reference composition is a composition ofoligonucleotides having the same base sequence and the same chemicalmodifications. In some embodiments, a reference composition is acomposition of oligonucleotides having the same base sequence and thesame pattern of chemical modifications. In some embodiments, a referencecomposition is a chirally un-controlled (or stereorandom) composition ofoligonucleotides having the same base sequence and chemicalmodifications.

In some embodiments, a reference composition is a composition ofoligonucleotides having the same base sequence but different chemicalmodifications. In some embodiments, a reference composition is acomposition of oligonucleotides having the same base sequence, basemodifications, internucleotidic linkage modifications but differentsugar modifications. In some embodiments, a reference composition hasfewer 2′-modified sugar modifications. In some embodiments, a referencecomposition is a composition of oligonucleotides having the same basesequence, base modifications, sugar modifications but differentinternucleotidic linkage modifications. In some embodiments, a referencecomposition has more internucleotidic linkage modifications. In someembodiments, a reference composition has fewer natural phosphatelinkages. In some embodiments, a reference composition comprisingoligonucleotides having no natural phosphate linkages.

In some embodiments, a reference composition is a composition comprisinga reference plurality of oligonucleotides wherein individualoligonucleotides within the reference plurality differ from one anotherin stereochemical structure. In some embodiments, a referencecomposition is a composition comprising a reference plurality ofoligonucleotides, wherein at least some oligonucleotides within thereference plurality have a structure different from a structurerepresented by a plurality of oligonucleotides of a composition comparedto the reference composition. In some embodiments, a referencecomposition is a composition comprising a reference plurality ofoligonucleotides wherein at least some oligonucleotides within thereference plurality do not comprise a wing region and a core region.

In some embodiments, a reference oligonucleotide composition comprises areference plurality of oligonucleotides having the same commonnucleotide sequence but lacking at least one of the one or more modifiedsugar moieties in oligonucleotides of the oligonucleotide compositioncompared to the reference composition. In some embodiments, a referenceoligonucleotide composition comprises a reference plurality ofoligonucleotides having the same common nucleotide sequence but have nomodified sugar moieties. In some embodiments, a referenceoligonucleotide composition comprises a reference plurality ofoligonucleotides having the same common nucleotide sequence but do notcomprise natural phosphate linkages. In some embodiments, a referencecomposition is a chirally controlled oligonucleotide composition ofoligonucleotides having the same chemical modification patterns. In someembodiments, a reference composition is a chirally controlledoligonucleotide composition of another stereoisomer.

In some embodiments, oligonucleotides of the first plurality compriseone or more structural elements (e.g., modifications, stereochemistry,patterns, etc.) that oligonucleotides of the reference plurality do notall have. Such structural elements can be any one described in thisdisclosure.

In some embodiments, oligonucleotides of the first plurality comprisemore phosphorothioate linkages than oligonucleotides of the referencecomposition. In some embodiments, oligonucleotides of the firstplurality comprise more phosphorothioate linkages than oligonucleotidesof the reference composition at the 5′-end region. In some embodiments,oligonucleotides of the first plurality comprise more phosphorothioatelinkages than oligonucleotides of the reference composition at the3′-end region. In some embodiments, oligonucleotides of the firstplurality comprise more phosphorothioate linkages in a wing region thanthe corresponding region of oligonucleotides of the referencecomposition. In some embodiments, oligonucleotides of the firstplurality comprise more phosphorothioate linkages in each wing regionthan the corresponding regions in oligonucleotides of the referencecomposition. In some embodiments, oligonucleotides of the firstplurality comprise more Sp chiral internucleotidic linkages thanoligonucleotides of the reference composition. In some embodiments,oligonucleotides of the first plurality comprise more Spphosphorothioate linkages than oligonucleotides of the referencecomposition. In some embodiments, oligonucleotides of the firstplurality comprise more Sp phosphorothioate linkages thanoligonucleotides of the reference composition at the 5′-end region. Insome embodiments, oligonucleotides of the first plurality comprise moreSp phosphorothioate linkages than oligonucleotides of the referencecomposition at the 3′-end region. In some embodiments, oligonucleotidesof the first plurality comprise more Sp phosphorothioate linkages in awing region than oligonucleotides of the reference composition. In someembodiments, oligonucleotides of the first plurality comprise more Spphosphorothioate linkages in each wing region than oligonucleotides ofthe reference composition. In some embodiments, oligonucleotides of thefirst plurality comprise more modified bases than oligonucleotides ofthe reference composition. In some embodiments, oligonucleotides of thefirst plurality comprise more methylated bases than oligonucleotides ofthe reference composition. In some embodiments, oligonucleotides of thefirst plurality comprise more methylated bases than oligonucleotides ofthe reference composition at the 5′-end region. In some embodiments,oligonucleotides of the first plurality comprise more methylated basesthan oligonucleotides of the reference composition at the 3′-end region.In some embodiments, oligonucleotides of the first plurality comprisemore methylated bases than in a wing region than oligonucleotides of thereference composition. In some embodiments, oligonucleotides of thefirst plurality comprise more methylated bases than in each wing regionthan oligonucleotides of the reference composition. In some embodiments,oligonucleotides of the first plurality comprise fewer 2′-MOEmodifications than oligonucleotides of the reference composition. Insome embodiments, oligonucleotides of the first plurality comprise fewer2′-MOE modifications than oligonucleotides of the reference composition.In some embodiments, oligonucleotides of the first plurality comprisefewer 2′-MOE modifications than oligonucleotides of the referencecomposition at the 5′-end region. In some embodiments, oligonucleotidesof the first plurality comprise fewer 2′-MOE modifications thanoligonucleotides of the reference composition at the 3′-end. In someembodiments, oligonucleotides of the first plurality comprise fewer2′-MOE modifications than in a wing region than oligonucleotides of thereference composition. In some embodiments, oligonucleotides of thefirst plurality comprise fewer 2′-MOE modifications than in each wingregion than oligonucleotides of the reference composition. In someembodiments, individual oligonucleotides within the reference pluralitydiffer from one another in stereochemical structure. In someembodiments, at least some oligonucleotides within the referenceplurality have a structure different from a structure represented by theplurality of oligonucleotides of the composition. In some embodiments,at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region. In some embodiments, thereference composition is a substantially racemic preparation ofoligonucleotides that share the base sequence. In some embodiments, thereference composition is a chirally controlled oligonucleotidecomposition of another oligonucleotide type. In some embodiments,oligonucleotides of the reference composition comprise morephosphorothioate linkages. In some embodiments, oligonucleotides of thereference composition comprise only phosphorothioate linkages. In someembodiments, oligonucleotides of the reference composition comprisefewer modified sugar moieties. In some embodiments, oligonucleotides ofthe reference composition comprise fewer modified sugar moieties,wherein the modification is 2′-OR¹. In some embodiments,oligonucleotides of the reference composition comprise more modifiedsugar moieties. In some embodiments, oligonucleotides of the referencecomposition comprise more modified sugar moieties, the modification is2′-OR¹. In some embodiments, oligonucleotides of the referencecomposition comprise fewer phosphorothioate linkages. In someembodiments, oligonucleotides of the reference composition have a wing,and comprise fewer phosphorothioate linkages at the wing. In someembodiments, oligonucleotides of the reference composition comprisefewer Sp phosphorothioate linkages. In some embodiments,oligonucleotides of the reference composition have a wing, and comprisefewer Sp phosphorothioate linkages at the wing. In some embodiments,oligonucleotides of the reference composition comprise more Rpphosphorothioate linkages. In some embodiments, oligonucleotides of thereference composition have a wing, and comprise more Rp phosphorothioatelinkages at the wing. In some embodiments, oligonucleotides of thereference composition comprise fewer methylated bases. In someembodiments, oligonucleotides of the reference composition comprise more2′-MOE modifications. In some embodiments, oligonucleotides of thereference composition comprise fewer natural phosphate linkages. In someembodiments, oligonucleotides of the reference composition comprisefewer natural phosphate linkages at the 5′- and/or 3′-end. In someembodiments, oligonucleotides of the reference composition comprisefewer natural phosphate linkages in a region corresponding to a wing ofoligonucleotides of the first plurality. In some embodiments,oligonucleotides of the first plurality comprise natural phosphatelinkages in a wing, and oligonucleotides of the reference compositioncomprise fewer natural phosphate linkages at the corresponding wingregion. In some embodiments, oligonucleotides of the first pluralitycomprises natural phosphate linkages in a wing, and oligonucleotides ofthe reference composition comprises modified internucleotidic linkagesat one or more such natural phosphate linkage locations in a wing. Insome embodiments, oligonucleotides of the first plurality comprisenatural phosphate linkages in a wing, and oligonucleotides of thereference composition comprises phosphorothioate linkages at one or moresuch natural phosphate linkage locations in a wing. In some embodiments,oligonucleotides of the reference composition comprise no naturalphosphate linkages. In some embodiments, oligonucleotides of thereference composition comprise no wing-core-wing structure. In someembodiments, oligonucleotides of the first plurality comprise a 5′-endwing region comprising a natural phosphate linkage between the twonucleosides at its 3′-end, and oligonucleotides of a reference pluralitydo not have a natural phosphate linkage at the same position. In someembodiments, oligonucleotides of the first plurality comprise a 3′-endwing region comprising a natural phosphate linkage between the twonucleosides at its 5′-end, and oligonucleotides of a reference pluralitydo not have a natural phosphate linkage at the same position.

In some embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of one oligonucleotide type. Insome embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of only one oligonucleotidetype. In some embodiments, provided chirally controlled oligonucleotidecompositions has oligonucleotides of only one oligonucleotide type. Insome embodiments, provided chirally controlled oligonucleotidecompositions comprises oligonucleotides of two or more oligonucleotidetypes. In some embodiments, using such compositions, provided methodscan target more than one target. In some embodiments, a chirallycontrolled oligonucleotide composition comprising two or moreoligonucleotide types targets two or more targets. In some embodiments,a chirally controlled oligonucleotide composition comprising two or moreoligonucleotide types targets two or more mismatches. In someembodiments, a single oligonucleotide type targets two or more targets,e.g., mutations. In some embodiments, a target region ofoligonucleotides of one oligonucleotide type comprises two or more“target sites” such as two mutations or SNPs.

In some embodiments, oligonucleotides in a provided chirally controlledoligonucleotide composition optionally comprise modified bases orsugars. In some embodiments, a provided chirally controlledoligonucleotide composition does not have any modified bases or sugars.In some embodiments, a provided chirally controlled oligonucleotidecomposition does not have any modified bases. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise modified bases and sugars. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise a modified base. In some embodiments,oligonucleotides in a provided chirally controlled oligonucleotidecomposition comprise a modified sugar. Modified bases and sugars foroligonucleotides are widely known in the art, including but not limitedin those described in the present disclosure. In some embodiments, amodified base is 5-mC. In some embodiments, a modified sugar is a2′-modified sugar. Suitable 2′-modification of oligonucleotide sugarsare widely known by a person having ordinary skill in the art. In someembodiments, 2′-modifications include but are not limited to 2′-OR¹,wherein R¹ is not hydrogen. In some embodiments, a 2′-modification is2′-OR¹, wherein R¹ is optionally substituted C₁₋₆ aliphatic. In someembodiments, a 2′-modification is 2′-MOE. In some embodiments, amodification is 2′-halogen. In some embodiments, a modification is 2′-F.In some embodiments, modified bases or sugars may further enhanceactivity, stability and/or selectivity of a chirally controlledoligonucleotide composition, whose common pattern of backbone chiralcenters provides unexpected activity, stability and/or selectivity.

In some embodiments, a provided chirally controlled oligonucleotidecomposition does not have any modified sugars. In some embodiments, aprovided chirally controlled oligonucleotide composition does not haveany 2′-modified sugars. In some embodiments, the present disclosuresurprisingly found that by using chirally controlled oligonucleotidecompositions, modified sugars are not needed for stability, activity,and/or control of cleavage patterns. Furthermore, in some embodiments,the present disclosure surprisingly found that chirally controlledoligonucleotide compositions of oligonucleotides without modified sugarsdeliver better properties in terms of stability, activity, turn-overand/or control of cleavage patterns. For example, in some embodiments,it is surprisingly found that chirally controlled oligonucleotidecompositions of oligonucleotides having no modified sugars dissociatesmuch faster from cleavage products and provide significantly increasedturn-over than compositions of oligonucleotides with modified sugars.

As discussed in detail herein, the present disclosure provides, amongother things, a chirally controlled oligonucleotide composition, meaningthat the composition contains a plurality of oligonucleotides of atleast one type. Each oligonucleotide molecule of a particular “type” iscomprised of preselected (e.g., predetermined) structural elements withrespect to: (1) base sequence; (2) pattern of backbone linkages; (3)pattern of backbone chiral centers; and (4) pattern of backboneP-modification moieties. In some embodiments, provided oligonucloetidecompositions contain oligonucleotides that are prepared in a singlesynthesis process. In some embodiments, provided compositions containoligonucloetides having more than one chiral configuration within asingle oligonucleotide molecule (e.g., where different residues alongthe oligonucleotide have different stereochemistry); in some suchembodiments, such oligonucleotides may be obtained in a single synthesisprocess, without the need for secondary conjugation steps to generateindividual oligonucleotide molecules with more than one chiralconfiguration.

Oligonucleotide compositions as provided herein can be used as agentsfor modulating a number of cellular processes and machineries, includingbut not limited to, transcription, translation, immune responses,epigenetics, etc. In addition, oligonucleotide compositions as providedherein can be used as reagents for research and/or diagnostic purposes.One of ordinary skill in the art will readily recognize that the presentdisclosure herein is not limited to particular use but is applicable toany situations where the use of synthetic oligonucleitides is desirable.Among other things, provided compositions are useful in a variety oftherapeutic, diagnostic, agricultural, and/or research applications.

In some embodiments, provided oligonucloetide compositions compriseoligonucleotides and/or residues thereof that include one or morestructural modifications as described in detail herein. In someembodiments, provided oligonucleotide compositions compriseoligonucleoties that contain one or more nucleic acid analogs. In someembodiments, provided oligonucleotide compositions compriseoligonucleotides that contain one or more artificial nucleic acids orresidues (e.g., a nucleotide analog), including but not limited to: apeptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino,threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleicacid (ANA), 2′-fluoroarabinose nucleic acid (FANA), cyclohexene nucleicacid (CeNA), anhydrohexitol nucleic acid (HNA), and/or unlocked nucleicacid (UNA), threose nucleic acids (TNA), and/or Xeno nucleic acids(XNA), and any combination thereof.

In any of the embodiments, the disclosure is useful foroligonucleotide-based modulation of gene expression, immune response,etc. Accordingly, stereo-defined, oligonucleotide compositions of thedisclosure, which contain oligonucleotides of predetermined type (i.e.,which are chirally controlled, and optionally chirally pure), can beused in lieu of conventional stereo-random or chirally impurecounterparts. In some embodiments, provided compositions show enhancedintended effects and/or reduced unwanted side effects. Certainembodiments of biological and clinical/therapeutic applications of thedisclosure are discussed explicitly below.

Various dosing regimens can be utilized to administer provided chirallycontrolled oligonucleotide compositions. In some embodiments, multipleunit doses are administered, separated by periods of time. In someembodiments, a given composition has a recommended dosing regimen, whichmay involve one or more doses. In some embodiments, a dosing regimencomprises a plurality of doses each of which are separated from oneanother by a time period of the same length; in some embodiments, adosing regimen comprises a plurality of doses and at least two differenttime periods separating individual doses. In some embodiments, all doseswithin a dosing regimen are of the same unit dose amount. In someembodiments, different doses within a dosing regimen are of differentamounts. In some embodiments, a dosing regimen comprises a first dose ina first dose amount, followed by one or more additional doses in asecond dose amount different from the first dose amount. In someembodiments, a dosing regimen comprises a first dose in a first doseamount, followed by one or more additional doses in a second (orsubsequent) dose amount that is same as or different from the first dose(or another prior dose) amount. In some embodiments, a dosing regimencomprises administering at least one unit dose for at least one day. Insome embodiments, a dosing regimen comprises administering more than onedose over a time period of at least one day, and sometimes more than oneday. In some embodiments, a dosing regimen comprises administeringmultiple doses over a time period of at least week. In some embodiments,the time period is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, adosing regimen comprises administering one dose per week for more thanone week. In some embodiments, a dosing regimen comprises administeringone dose per week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, a dosingregimen comprises administering one dose every two weeks for more thantwo week period. In some embodiments, a dosing regimen comprisesadministering one dose every two weeks over a time period of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more(e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more)weeks. In some embodiments, a dosing regimen comprises administering onedose per month for one month. In some embodiments, a dosing regimencomprises administering one dose per month for more than one month. Insome embodiments, a dosing regimen comprises administering one dose permonth for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In someembodiments, a dosing regimen comprises administering one dose per weekfor about 10 weeks. In some embodiments, a dosing regimen comprisesadministering one dose per week for about 20 weeks. In some embodiments,a dosing regimen comprises administering one dose per week for about 30weeks. In some embodiments, a dosing regimen comprises administering onedose per week for 26 weeks. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that differs from that utilized for a chirally uncontrolled(e.g., stereorandom) oligonucleotide composition of the same sequence,and/or of a different chirally controlled oligonucleotide composition ofthe same sequence. In some embodiments, a chirally controlledoligonucleotide composition is administered according to a dosingregimen that is reduced as compared with that of a chirally uncontrolled(e.g., sterorandom) oligonucleotide composition of the same sequence inthat it achieves a lower level of total exposure over a given unit oftime, involves one or more lower unit doses, and/or includes a smallernumber of doses over a given unit of time. In some embodiments, achirally controlled oligonucleotide composition is administeredaccording to a dosing regimen that extends for a longer period of timethan does that of a chirally uncontrolled (e.g., stereorandom)oligonucleotide composition of the same sequence Without wishing to belimited by theory, Applicant notes that in some embodiments, the shorterdosing regimen, and/or longer time periods between doses, may be due tothe improved stability, bioavailability, and/or efficacy of a chirallycontrolled oligonucleotide composition. In some embodiments, a chirallycontrolled oligonucleotide composition has a longer dosing regimencompared to the corresponding chirally uncontrolled oligonucleotidecomposition. In some embodiments, a chirally controlled oligonucleotidecomposition has a shorter time period between at least two dosescompared to the corresponding chirally uncontrolled oligonucleotidecomposition. Without wishing to be limited by theory, Applicant notesthat in some embodiments longer dosing regimen, and/or shorter timeperiods between doses, may be due to the improved safety of a chirallycontrolled oligonucleotide composition.

In some embodiments, with their low toxicity, provided oligonucleotidesand compositions can be administered in higher dosage and/or with higherfrequency. In some embodiments, with their improved delivery (and otherproperties), provided compositions can be administered in lower dosagesand/or with lower frequency to achieve biological effects, for example,clinical efficacy.

A single dose can contain various amounts of oligonucleotides. In someembodiments, a single dose can contain various amounts of a type ofchirally controlled oligonucleotide, as desired suitable by theapplication. In some embodiments, a single dose contains about 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more(e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000 or more) mg of a type of chirally controlled oligonucleotide.In some embodiments, a single dose contains about 1 mg of a type ofchirally controlled oligonucleotide. In some embodiments, a single dosecontains about 5 mg of a type of chirally controlled oligonucleotide. Insome embodiments, a single dose contains about 10 mg of a type ofchirally controlled oligonucleotide. In some embodiments, a single dosecontains about 15 mg of a type of chirally controlled oligonucleotide.In some embodiments, a single dose contains about 20 mg of a type ofchirally controlled oligonucleotide. In some embodiments, a single dosecontains about 50 mg of a type of chirally controlled oligonucleotide.In some embodiments, a single dose contains about 100 mg of a type ofchirally controlled oligonucleotide. In some embodiments, a single dosecontains about 150 mg of a type of chirally controlled oligonucleotide.In some embodiments, a single dose contains about 200 mg of a type ofchirally controlled oligonucleotide. In some embodiments, a single dosecontains about 250 mg of a type of chirally controlled oligonucleotide.In some embodiments, a single dose contains about 300 mg of a type ofchirally controlled oligonucleotide. In some embodiments, a chirallycontrolled oligonucleotide is administered at a lower amount in a singledose, and/or in total dose, than a chirally uncontrolledoligonucleotide. In some embodiments, a chirally controlledoligonucleotide is administered at a lower amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide dueto improved efficacy. In some embodiments, a chirally controlledoligonucleotide is administered at a higher amount in a single dose,and/or in total dose, than a chirally uncontrolled oligonucleotide. Insome embodiments, a chirally controlled oligonucleotide is administeredat a higher amount in a single dose, and/or in total dose, than achirally uncontrolled oligonucleotide due to improved safety.

Biologically Active Oligonucleotides

A provided oligonucleotide composition as used herein may comprisesingle stranded and/or multiply stranded oligonucleotides. In someembodiments, single-stranded oligonucleotides contain self-complementaryportions that may hybridize under relevant conditions so that, as used,even single-stranded oligonucleotides may have at least partiallydouble-stranded character. In some embodiments, an oligonucleotideincluded in a provided composition is single-stranded, double-stranded,or triple-stranded. In some embodiments, an oligonucleotide included ina provided composition comprises a single-stranded portion and amultiple-stranded portion within the oligonucleotide. In someembodiments, as noted above, individual single-stranded oligonucleotidescan have double-stranded regions and single-stranded regions.

In some embodiments, provided compositions include one or moreoligonucleotides fully or partially complementary to strand of:structural genes, genes control and/or termination regions, and/orself-replicating systems such as viral or plasmid DNA. In someembodiments, provided compositions include one or more oligonucleotidesthat are or act as siRNAs or other RNA interference reagents (RNAiagents or iRNA agents), shRNA, antisense oligonucleotides, self-cleavingRNAs, ribozymes, fragment thereof and/or variants thereof (such asPeptidyl transferase 23S rRNA, RNase P, Group I and Group II introns,GIR1 branching ribozymes, Leadzyme, Hairpin ribozymes, Hammerheadribozymes, HDV ribozymes, Mammalian CPEB3 ribozyme, VS ribozymes, glmSribozymes, CoTC ribozyme, etc.), microRNAs, microRNA mimics, supermirs,aptamers, antimirs, antagomirs, U1 adaptors, triplex-formingoligonucleotides, RNA activators, long non-coding RNAs, short non-codingRNAs (e.g., piRNAs), immunomodulatory oligonucleotides (such asimmunostimulatory oligonucleotides, immunoinhibitory oligonucleotides),GNA, LNA, ENA, PNA, TNA, HNA, TNA, XNA, HeNA, CeNA, morpholinos,G-quadruplex (RNA and DNA), antiviral oligonucleotides, and decoyoligonucleotides.

In some embodiments, provided compositions include one or more hybrid(e.g., chimeric) oligonucleotides. In the context of the presentdisclosure, the term “hybrid” broadly refers to mixed structuralcomponents of oligonucloetides. Hybrid oligonucleotides may refer to,for example, (1) an oligonucleotide molecule having mixed classes ofnucleotides, e.g., part DNA and part RNA within the single molecule(e.g., DNA-RNA); (2) complementary pairs of nucleic acids of differentclasses, such that DNA:RNA base pairing occurs either intramolecularlyor intermolecularly; or both; (3) an oligonucleotide with two or morekinds of the backbone or internucleotide linkages.

In some embodiments, provided compositions include one or moreoligonucleotide that comprises more than one classes of nucleic acidresidues within a single molecule. For example, in any of theembodiments described herein, an oligonucleotide may comprise a DNAportion and an RNA portion. In some embodiments, an oligonucleotide maycomprise a unmodified portion and modified portion.

Provided oligonucleotide compositions can include oligonucleotidescontaining any of a variety of modifications, for example as describedherein. In some embodiments, particular modifications are selected, forexample, in light of intended use. In some embodiments, it is desirableto modify one or both strands of a double-stranded oligonucleotide (or adouble-stranded portion of a single-stranded oligonucleotide). In someembodiments, the two strands (or portions) include differentmodifications. In some embodiments, the two strands include the samemodifications. One of skill in the art will appreciate that the degreeand type of modifications enabled by methods of the present disclosureallow for numerous permutations of modifications to be made. Examplesuch modifications are described herein and are not meant to belimiting.

The phrase “antisense strand” as used herein, refers to anoligonucleotide that is substantially or 100% complementary to a targetsequence of interest. The phrase “antisense strand” includes theantisense region of both oligonucleotides that are formed from twoseparate strands, as well as unimolecular oligonucleotides that arecapable of forming hairpin or dumbbell type structures. The terms“antisense strand” and “guide strand” are used interchangeably herein.

The phrase “sense strand” refers to an oligonucleotide that has the samenucleoside sequence, in whole or in part, as a target sequence such as amessenger RNA or a sequence of DNA. The terms “sense strand” and“passenger strand” are used interchangeably herein.

By “target sequence” is meant any nucleic acid sequence whose expressionor activity is to be modulated. The target nucleic acid can be DNA orRNA, such as endogenous DNA or RNA, viral DNA or viral RNA, or other RNAencoded by a gene, virus, bacteria, fungus, mammal, or plant. In someembodiments, a target sequence is associated with a disease or disorder.

By “specifically hybridizable” and “complementary” is meant that anucleic acid can form hydrogen bond(s) with another nucleic acidsequence by either traditional Watson-Crick or other non-traditionaltypes. In reference to the nucleic molecules of the present disclosure,the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LIT pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, /. Ain. Chem. Soc. 109:3783-3785)

A percent complementarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” or 100% complementarity meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. Less than perfect complementarity refers to thesituation in which some, but not all, nucleoside units of two strandscan hydrogen bond with each other. “Substantial complementarity” refersto polynucleotide strands exhibiting 90% or greater complementarity,excluding regions of the polynucleotide strands, such as overhangs, thatare selected so as to be noncomplementary. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, e.g., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.In some embodiments, non-target sequences differ from correspondingtarget sequences by at least 5 nucleotides.

When used as therapeutics, a provided oligonucleotide is administered asa pharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprises a therapeutically effective amount of a providedoligonucleotide comprising, or a pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable inactiveingredient selected from pharmaceutically acceptable diluents,pharmaceutically acceptable excipients, and pharmaceutically acceptablecarriers. In another embodiment, the pharmaceutical composition isformulated for intravenous injection, oral administration, buccaladministration, inhalation, nasal administration, topicaladministration, ophthalmic administration or otic administration. Infurther embodiments, the pharmaceutical composition is a tablet, a pill,a capsule, a liquid, an inhalant, a nasal spray solution, a suppository,a suspension, a gel, a colloid, a dispersion, a suspension, a solution,an emulsion, an ointment, a lotion, an eye drop or an ear drop.

Pharmaceutical Compositions

When used as therapeutics, a provided oligonucleotide or oligonucleotidecomposition described herein is administered as a pharmaceuticalcomposition. In some embodiments, the pharmaceutical compositioncomprises a therapeutically effective amount of a providedoligonucleotides, or a pharmaceutically acceptable salt thereof, and atleast one pharmaceutically acceptable inactive ingredient selected frompharmaceutically acceptable diluents, pharmaceutically acceptableexcipients, and pharmaceutically acceptable carriers. In someembodiments, the pharmaceutical composition is formulated forintravenous injection, oral administration, buccal administration,inhalation, nasal administration, topical administration, ophthalmicadministration or otic administration. In some embodiments, thepharmaceutical composition is a tablet, a pill, a capsule, a liquid, aninhalant, a nasal spray solution, a suppository, a suspension, a gel, acolloid, a dispersion, a suspension, a solution, an emulsion, anointment, a lotion, an eye drop or an ear drop.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising chirally controlled oligonucleotide, orcomposition thereof, in admixture with a pharmaceutically acceptableexcipient. One of skill in the art will recognize that thepharmaceutical compositions include the pharmaceutically acceptablesalts of the chirally controlled oligonucleotide, or compositionthereof, described above.

A variety of supramolecular nanocarriers can be used to deliver nucleicacids. Example nanocarriers include, but are not limited to liposomes,cationic polymer complexes and various polymeric. Complexation ofnucleic acids with various polycations is another approach forintracellular delivery; this includes use of PEGlyated polycations,polyethyleneamine (PEI) complexes, cationic block co-polymers, anddendrimers. Several cationic nanocarriers, including PEI andpolyamidoamine dendrimers help to release contents from endosomes. Otherapproaches include use of polymeric nanoparticles, polymer micelles,quantum dots and lipoplexes.

Additional nucleic acid delivery strategies are known in addition to theexample delivery strategies described herein.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington, The Science andPractice of Pharmacy, (20th ed. 2000).

Provided oligonucleotides, and compositions thereof, are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from about 0.01 to about 1000 mg, from about 0.5 to about 100mg, from about 1 to about 50 mg per day, and from about 5 to about 100mg per day are examples of dosages that may be used. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, and the preference and experience of theattending physician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington, The Science and Practice of Pharmacy (20th ed. 2000).Preferred pharmaceutically acceptable salts include, for example,acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide,hydrochloride, maleate, mesylate, napsylate, pamoate (embonate),phosphate, salicylate, succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington, The Science and Practice of Pharmacy (20th ed. 2000).Suitable routes may include oral, buccal, by inhalation spray,sublingual, rectal, transdermal, vaginal, transmucosal, nasal orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articullar, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections or other modes ofdelivery.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions, such as in physiologically compatiblebuffers such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection.

The compounds can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds of the disclosure tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject(e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure may alsobe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

In certain embodiments, oligonucleotides and compositions are deliveredto the CNS. In certain embodiments, oligonucleotides and compositionsare delivered to the cerebrospinal fluid. In certain embodiments,oligonucleotides and compositions are administered to the brainparenchyma. In certain embodiments, oligonucleotides and compositionsare delivered to an animal/subject by intrathecal administration, orintracerebroventricular administration. Broad distribution ofoligonucleotides and compositions, described herein, within the centralnervous system may be achieved with intraparenchymal administration,intrathecal administration, or intracerebroventricular administration.

In certain embodiments, parenteral administration is by injection, by,e.g., a syringe, a pump, etc. In certain embodiments, the injection is abolus injection. In certain embodiments, the injection is administereddirectly to a tissue, such as striatum, caudate, cortex, hippocampus andcerebellum.

In certain embodiments, methods of specifically localizing apharmaceutical agent, such as by bolus injection, decreases medianeffective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or50. In certain embodiments, the pharmaceutical agent in an antisensecompound as further described herein. In certain embodiments, thetargeted tissue is brain tissue. In certain embodiments the targetedtissue is striatal tissue. In certain embodiments, decreasing EC50 isdesirable because it reduces the dose required to achieve apharmacological result in a patient in need thereof.

In certain embodiments, an antisense oligonucleotide is delivered byinjection or infusion once every month, every two months, every 90 days,every 3 months, every 6 months, twice a year or once a year.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with oligonucleotides of this disclosure. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with the oligonucleotides of this disclosure to treatproliferative diseases and cancer. Examples of known chemotherapeuticagents include, but are not limited to, adriamycin, dexamethasone,vincristine, cyclophosphamide, fluorouracil, topotecan, taxol,interferons, and platinum derivatives.

The function and advantage of these and other embodiments of the presentdisclosure will be more fully understood from the examples describedbelow. The following examples are intended to illustrate the benefits ofthe present disclosure, but do not exemplify the full scope of thedisclosure.

Lipids

In some embodiments, provided oligonucleotide compositions furthercomprise one or more lipids. In some embodiments, the lipids areconjugated to provided oligonucleotides in the compositions. In someembodiments, two or more same or different lipids can be conjugated toone oligonucleotide, through either the same or differently chemistryand/or locations. In some embodiments, a composition can comprise anoligonucleotide disclosed herein (as non-limiting examples, a chirallycontrolled oligonucleotide composition, or a chirally controlledoligonucleotide composition wherein the sequence of the oligonucleotidecomprises, consists of or is the sequence of any oligonucleotidedisclosed herein, or a chirally controlled oligonucleotide compositionwherein the sequence of the oligonucleotide comprises, consists of or isthe sequence of any oligonucleotide disclosed in Table 8 or any otherTable herein, etc.) and a lipid. In some embodiments, a providedoligonucleotide comprises base sequence, pattern of backbone linkages,pattern or backbone chiral centers, and/or pattern of chemicalmodifications (e.g., base modifications, sugar modifications, etc.) ofany oligonucleotide disclosed herein, and is conjugated to a lipid. Insome embodiments, a provided composition comprises an oligonucleotidedisclosed herein and a lipid, wherein the lipid is conjugated to theoligonucleotide.

In some embodiments, the present disclosure provides a compositioncomprising an oligonucleotide amd a lipid. Many lipids can be utilizedin provided technologies in accordance with the present disclosure.

In some embodiments, a lipid comprises an R^(LD) group, wherein R^(LD)is an optionally substituted, C₁₀-C₅₀ saturated or partially unsaturatedaliphatic group, wherein one or more methylene units are optionally andindependently replaced by an optionally substituted group selected fromC₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—,and —C(O)O—, wherein: each R′ is independently —R, —C(O)R, —CO₂R, or—SO₂R, or:

-   -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;

-   -Cy- is an optionally substituted bivalent ring selected from    carbocyclylene, arylene, heteroarylene, and heterocyclylene; and

-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl.

In some embodiments, a lipid comprises an R^(LD) group, wherein R^(LD)is an optionally substituted, C₁₀-C₆₀ saturated or partially unsaturatedaliphatic group, wherein one or more methylene units are optionally andindependently replaced by an optionally substituted group selected fromC₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C≡, a C₁-C₆ heteroaliphatic moiety,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—,and —C(O)O—, wherein:

-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    carbocyclylene, arylene, heteroarylene, and heterocyclylene; and-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl.

In some embodiments, a lipid comprises an R^(LD) group, wherein R^(LD)is an optionally substituted, C₁₀-C₄₀ saturated or partially unsaturatedaliphatic group, wherein one or more methylene units are optionally andindependently replaced by an optionally substituted group selected fromC₁-C₆ alkylene, C₁-C₆ alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety,—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—,and —C(O)O—, wherein:

-   each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:    -   two R′ are taken together with their intervening atoms to form        an optionally substituted aryl, carbocyclic, heterocyclic, or        heteroaryl ring;-   -Cy- is an optionally substituted bivalent ring selected from    carbocyclylene, arylene, heteroarylene, and heterocyclylene; and-   each R is independently hydrogen, or an optionally substituted group    selected from C₁-C₆ aliphatic, phenyl, carbocyclyl, aryl,    heteroaryl, or heterocyclyl.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₈₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is a hydrocarbon group consisting carbon andhydrogen atoms.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is a hydrocarbon group consisting carbon andhydrogen atoms.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₄₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or moremethylene units are optionally and independently replaced by anoptionally substituted group selected from C₁-C₆ alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆ heteroaliphatic moiety, —C(R′)₂—, and -Cy-.In some embodiments, R^(LD) is a hydrocarbon group consisting carbon andhydrogen atoms.

The aliphatic group of R^(LD) can be a variety of suitable length. Insome embodiments, it is C₁₀-C₈₀. In some embodiments, it is C₁₀-C₇₅. Insome embodiments, it is C₁₀-C₇₀. In some embodiments, it is C₁₀-C₆₅. Insome embodiments, it is C₁₀-C₆₀. In some embodiments, it is C₁₀-C₅₀. Insome embodiments, it is C₁₀-C₄₀. In some embodiments, it is C₁₀-C₃₅. Insome embodiments, it is C₁₀-C₃₀. In some embodiments, it is C₁₀-C₂₅. Insome embodiments, it is C₁₀-C₂₄. In some embodiments, it is C₁₀-C₂₃. Insome embodiments, it is C₁₀-C₂₂. In some embodiments, it is C₁₀-C₂₁. Insome embodiments, it is C₁₂-C₂₂. In some embodiments, it is C₁₃-C₂₂. Insome embodiments, it is C₁₄-C₂₂. In some embodiments, it is C₁₅-C₂₂. Insome embodiments, it is C₁₆-C₂₂. In some embodiments, it is C₁₇-C₂₂. Insome embodiments, it is C₁₈-C₂₂. In some embodiments, it is C₁₀-C₂₀. Insome embodiments, the lower end of the range is C₁₀, C₁₁, C₁₂, C₁₃, C₁₄,C₁₅, C₁₆, C₁₇, or C₁₈. In some embodiments, the higher end of the rangeis Cis, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₅,C₄₀, C₄₅, C₅₀, C₅₅, or C₆₀. In some embodiments, it is C₁₀. In someembodiments, it is C₁₁. In some embodiments, it is C₁₂. In someembodiments, it is C₁₃. In some embodiments, it is C₁₄. In someembodiments, it is C₁₅. In some embodiments, it is C₁₆. In someembodiments, it is C₁₇. In some embodiments, it is Cis. In someembodiments, it is C₁₉. In some embodiments, it is C₂₀. In someembodiments, it is C₂₁. In some embodiments, it is C₂₂. In someembodiments, it is C₂₃. In some embodiments, it is C₂₄. In someembodiments, it is C₂₅. In some embodiments, it is C₃₀. In someembodiments, it is C₃₅. In some embodiments, it is C₄₀. In someembodiments, it is C₄₅. In some embodiments, it is C₅₀. In someembodiments, it is C₅₅. In some embodiments, it is C₆₀.

In some embodiments, a lipid comprises no more than one R^(LD) group. Insome embodiments, a lipid comprises two or more R^(LD) groups.

In some embodiments, a lipid is conjugated to a biologically activeagent, optionally through a linker, as a moiety comprising an R^(LD)group. In some embodiments, a lipid is conjugated to a biologicallyactive agent, optionally through a linker, as a moiety comprising nomore than one R^(LD) group. In some embodiments, a lipid is conjugatedto a biologically active agent, optionally through a linker, as anR^(LD) group. In some embodiments, a lipid is conjugated to abiologically active agent, optionally through a linker, as a moietycomprising two or more R^(LD) groups.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₄₀saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₄₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is a C₁₀-C₄₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁₋₄ aliphatic groups. In some embodiments, a lipid comprises a C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₄ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more C₁₋₂ aliphaticgroups. In some embodiments, a lipid comprises a C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁₋₂ aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more methyl groups.In some embodiments, a lipid comprises a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more methyl groups.

In some embodiments, R^(LD) is an unsubstituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an unsubstituted C₁₀-C₄₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises no more than one optionallysubstituted C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain. In some embodiments, a lipid comprises two or moreoptionally substituted C₁₀-C₄₀ linear, saturated or partiallyunsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₆₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is a C₁₀-C₆₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁-4 aliphatic groups. In some embodiments, a lipid comprises a C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁-4 aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₆₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more C₁-2 aliphaticgroups. In some embodiments, a lipid comprises a C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁-2 aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₆₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more methyl groups.In some embodiments, a lipid comprises a C₁₀-C₆₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more methyl groups.

In some embodiments, R^(LD) is an unsubstituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an unsubstituted C₁₀-C₆₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises no more than one optionallysubstituted C₁₀-C₆₀ linear, saturated or partially unsaturated,aliphatic chain. In some embodiments, a lipid comprises two or moreoptionally substituted C₁₀-C₆₀ linear, saturated or partiallyunsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted, C₁₀-C₈₀saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₈₀saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is an optionally substituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an optionally substituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, R^(LD) is a C₁₀-C₈₀ linear, saturated or partiallyunsaturated, aliphatic chain, optionally substituted with one or moreC₁-4 aliphatic groups. In some embodiments, a lipid comprises a C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁-4 aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₈₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more C₁-2 aliphaticgroups. In some embodiments, a lipid comprises a C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain, optionallysubstituted with one or more C₁-2 aliphatic groups. In some embodiments,R^(LD) is a C₁₀-C₈₀ linear, saturated or partially unsaturated,aliphatic chain, optionally substituted with one or more methyl groups.In some embodiments, a lipid comprises a C₁₀-C₈₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more methyl groups.

In some embodiments, R^(LD) is an unsubstituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain. In someembodiments, a lipid comprises an unsubstituted C₁₀-C₈₀ linear,saturated or partially unsaturated, aliphatic chain.

In some embodiments, a lipid comprises no more than one optionallysubstituted C₁₀-C₈₀ linear, saturated or partially unsaturated,aliphatic chain. In some embodiments, a lipid comprises two or moreoptionally substituted C₁₀-C₈₀ linear, saturated or partiallyunsaturated, aliphatic chain.

In some embodiments, R^(LD) is or comprises a C₁₀ saturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₁₀partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₁ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₁₁ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₁₂saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₂ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₁₃ saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₁₃ partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₄ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₄ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a Cis saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aCis partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₆ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₁₆ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₁₇saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₇ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a Cis saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a Cis partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₁₉ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₁₉ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₀ saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aC₂₀ partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₁ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₁ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₂₂saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₂ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₃ saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₃ partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₄ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₄ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₅ saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aC₂₅ partially unsaturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₆ saturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₆ partially unsaturated linearaliphatic chain. In some embodiments, R^(LD) is or comprises a C₂₇saturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₇ partially unsaturated linear aliphatic chain. In someembodiments, R^(LD) is or comprises a C₂₈ saturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₂₈ partiallyunsaturated linear aliphatic chain. In some embodiments, R^(LD) is orcomprises a C₂₉ saturated linear aliphatic chain. In some embodiments,R^(LD) is or comprises a C₂₉ partially unsaturated linear aliphaticchain. In some embodiments, R^(LD) is or comprises a C₃₀ saturatedlinear aliphatic chain. In some embodiments, R^(LD) is or comprises aC₃₀ partially unsaturated linear aliphatic chain.

In some embodiments, a lipid has the structure of R^(LD)—OH. In someembodiments, a lipid has the structure of R^(LD)—C(O)OH. In someembodiments, R^(LD) is

In some embodiments, a lipid is lauric acid, myristic acid, palmiticacid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid,gamma-linolenic acid, docosahexaenoic acid (DHA or cis-DHA), turbinaricacid, arachidonic acid, and dilinoleyl. In some embodiments, a lipid islauric acid, myristic acid, palmitic acid, stearic acid, oleic acid,linoleic acid, alpha-linolenic acid, gamma-linolenic acid,docosahexaenoic acid (DHA or cis-DHA), turbinaric acid, and dilinoleyl.In some embodiments, a lipid has a structure of:

In some embodiments, a lipid is, comprises or consists of any of: an atleast partially hydrophobic or amphiphilic molecule, a phospholipid, atriglyceride, a diglyceride, a monoglyceride, a fat-soluble vitamin, asterol, a fat and a wax. In some embodiments, a lipid is any of: a fattyacid, glycerolipid, glycerophospholipid, sphingolipid, sterol lipid,prenol lipid, saccharolipid, polyketide, and other molecule.

Lipids can be incorporated into provided technologies through many typesof methods in accordance with the present disclosure. In someembodiments, lipids are physically mixed with provided oligonucleotidesto form provided compositions. In some embodiments, lipids arechemically conjugated with oligonucleotides.

In some embodiments, provided compositions comprise two or more lipids.In some embodiments, provided oligonucleotides comprise two or moreconjugated lipids. In some embodiments, the two or more conjugatedlipids are the same. In some embodiments, the two or more conjugatedlipids are different. In some embodiments, provided oligonucleotidescomprise no more than one lipid. In some embodiments, oligonucleotidesof a provided composition comprise different types of conjugated lipids.In some embodiments, oligonucleotides of a provided composition comprisethe same type of lipids.

Lipids can be conjugated to oligonucleotides optionally through linkers.Various types of linkers in the art can be utilized in accordance of thepresent disclosure. In some embodiments, a linker comprise a phosphategroup, which can, for example, be used for conjugating lipids throughchemistry similar to those employed in oligonucleotide synthesis. Insome embodiments, a linker comprises an amide, ester, or ether group. Insome embodiments, a linker has the structure of -L-. In someembodiments, after conjugation to oligonucleotides, a lipid forms amoiety having the structure of -L-R^(LD), wherein each of L and R^(LD)is independently as defined and described herein.

In some embodiments, -L- comprises a bivalent aliphatic chain. In someembodiments, -L- comprises a phosphate group. In some embodiments, -L-comprises a phosphorothioate group. In some embodiments, -L- has thestructure of —C(O)NH—(CH₂)₆—OP(═O)(S—)—.

Lipids, optionally through linkers, can be conjugated tooligonucleotides at various suitable locations. In some embodiments,lipids are conjugated through the 5′-OH group. In some embodiments,lipids are conjugated through the 3′-OH group. In some embodiments,lipids are conjugated through one or more sugar moieties. In someembodiments, lipids are conjugated through one or more bases. In someembodiments, lipids are incorporated through one or moreinternucleotidic linkages. In some embodiments, an oligonucleotide maycontain multiple conjugated lipids which are independently conjugatedthrough its 5′-OH, 3′-OH, sugar moieties, base moieties and/orinternucleotidic linkages.

In some embodiments, a lipid is conjugated to an oligonucleotideoptionally through a linker moiety. A person having ordinary skill inthe art appreciates that various technologies can be utilized toconjugate lipids to an oligonucleotide in accordance with the presentdisclosure. For example, for lipids comprising carboxyl groups, suchlipids can be conjugated through the carboxyl groups. In someembodiments, a lipid is conjugated through a linker having the structureof -L-, wherein L is as defined and described in formula I. In someembodiments, L comprises a phosphate diester or modified phosphatediester moiety. In some embodiments, a compound formed by lipidconjugation has the structure of (R^(L)D-L-)_(x)-(oligonucleotide),wherein x is 1 or an integer greater than 1, and each of R^(LD) and L isindependently as defined and described herein. In some embodiments, xis 1. In some embodiments, x is greater than 1. In some embodiments, anoligonucleotide is an oligonucleotide. For example, in some embodiments,a conjugate has the following structures:

In some embodiments, a linker is selected from: an uncharged linker; acharged linker; a linker comprising an alkyl; a linker comprising aphosphate; a branched linker; an unbranched linker; a linker comprisingat least one cleavage group; a linker comprising at least one redoxcleavage group; a linker comprising at least one phosphate-basedcleavage group; a linker comprising at least one acid-cleavage group; alinker comprising at least one ester-based cleavage group; and a linkercomprising at least one peptide-based cleavage group.

In some embodiments, a lipid is not conjugated to an oligonucleotide.

In some embodiments, the present disclosure pertains to compositions andmethods related to a composition comprising an oligonucleotide and alipid comprising a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, wherein the lipid is conjugated to the biologicallyactive agent. In some embodiments, the present disclosure pertains tocompositions and methods related to a composition comprising anoligonucleotide and a lipid comprising a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more C₁₋₄ aliphatic group, wherein the lipid is conjugated to thebiologically active agent.

In some embodiments, the present disclosure pertains to compositions andmethods related to a composition comprising an oligonucleotide and alipid comprising a C₁₀-C₄₀ linear, saturated or partially unsaturated,aliphatic chain, wherein the lipid is not conjugated to the biologicallyactive agent. In some embodiments, the present disclosure pertains tocompositions and methods related to a composition comprising anoligonucleotide and a lipid comprising a C₁₀-C₄₀ linear, saturated orpartially unsaturated, aliphatic chain, optionally substituted with oneor more C₁₋₄ aliphatic group, wherein the lipid is not conjugated to thebiologically active agent.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is not conjugated to the biologicallyactive agent. In some embodiments, a composition comprises anoligonucleotide and a lipid selected from: lauric acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenicacid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaricacid, and dilinoleyl, wherein the lipid is not conjugated to thebiologically active agent.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is conjugated to the biologicallyactive agent. In some embodiments, a composition comprises anoligonucleotide and a lipid selected from: lauric acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenicacid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaricacid, and dilinoleyl, wherein the lipid is conjugated to thebiologically active agent.

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is directly conjugated to thebiologically active agent (without a linker interposed between the lipidand the biologically active agent). In some embodiments, a compositioncomprises an oligonucleotide and a lipid selected from: lauric acid,myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid,alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid(cis-DHA), turbinaric acid, and dilinoleyl, wherein the lipid isdirectly conjugated to the biologically active agent (without a linkerinterposed between the lipid and the biologically active agent).

In some embodiments, a composition comprises an oligonucleotide and alipid selected from: lauric acid, myristic acid, palmitic acid, stearicacid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenicacid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid,and dilinoleyl, wherein the lipid is indirectly conjugated to thebiologically active agent (with a linker interposed between the lipidand the biologically active agent). In some embodiments, a compositioncomprises an oligonucleotide and a lipid selected from: lauric acid,myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid,alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid(cis-DHA), turbinaric acid, and dilinoleyl, wherein the lipid isindirectly conjugated to the biologically active agent (with a linkerinterposed between the lipid and the biologically active agent).

A linker is a moiety that connects two parts of a composition; as anon-limiting example, a linker physically connects an oligonucleotide toa lipid.

Non-limiting examples of suitable linkers include: an uncharged linker;a charged linker; a linker comprising an alkyl; a linker comprising aphosphate; a branched linker; an unbranched linker; a linker comprisingat least one cleavage group; a linker comprising at least one redoxcleavage group; a linker comprising at least one phosphate-basedcleavage group; a linker comprising at least one acid-cleavage group; alinker comprising at least one ester-based cleavage group; a linkercomprising at least one peptide-based cleavage group.

In some embodiments, a linker comprises an uncharged linker or a chargedlinker.

In some embodiments, a linker comprises an alkyl.

In some embodiments, a linker comprises a phosphate. In variousembodiments, a phosphate can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon can be done. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen can be done. In variousembodiments, the linker comprising a phosphate comprises any one or moreof: a phosphorodithioate, phosphoramidate, boranophosphonoate, or acompound of formula (I):

where R³ is selected from OH, SH, NH₂, BH₃, CH₃, C₁₋₆ alkyl, C₆₋₁₀ aryl,C₁₋₆ alkoxy and C₆₋io aryloxy, wherein C₁₋₆ alkyl and C₆₋₁₀ aryl areunsubstituted or optionally independently substituted with 1 to 3 groupsindependently selected from halo, hydroxyl and NH₂; and R⁴ is selectedfrom O, S, NH, or CH₂.

In some embodiments, a linker comprises a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroaryl alkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R₁)₂,C(O), cleavable linking group, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclic; where R¹ is hydrogen, acyl, aliphatic or substitutedaliphatic.

In some embodiments, a linker is a branched linker. In some embodiments,a branchpoint of the branched linker may be at least trivalent, but maybe a tetravalent, pentavalent or hexavalent atom, or a group presentingsuch multiple valencies. In some embodiments, a branchpoint is —N,—N(Q)-C, —O—C, —S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or—N(Q)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

In one embodiment, a linker comprises at least one cleavable linkinggroup.

As a non-limiting example, a cleavable linking group can be sufficientlystable outside the cell, but which upon entry into a target cell iscleaved to release the two parts the linker is holding together. As anon-limiting example, a cleavable linking group is cleaved at least 10times or more, at least 100 times faster in the target cell or under afirst reference condition (which can, e.g., be selected to mimic orrepresent intracellular conditions) than in the blood of a subject, orunder a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum). Cleavablelinking groups are susceptible to cleavage agents, e.g., pH, redoxpotential or the presence of degradative molecules. Generally, cleavageagents are more prevalent or found at higher levels or activities insidecells than in serum or blood. Examples of such degradative agentsinclude: redox agents which are selected for particular substrates orwhich have no substrate specificity, including, e.g., oxidative orreductive enzymes or reductive agents such as mercaptans, present incells, that can degrade a redox cleavable linking group by reduction;esterases; endosomes or agents that can create an acidic environment,e.g., those that result in a pH of five or lower; enzymes that canhydrolyze or degrade an acid cleavable linking group by acting as ageneral acid, peptidases (which can be substrate specific), andphosphatases.

As a non-limiting example, a cleavable linkage group, such as adisulfide bond can be susceptible to pH. The pH of human serum is 7.4,while the average intracellular pH is slightly lower, ranging from about7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, andlysosomes have an even more acidic pH at around 5.0. Some linkers willhave a cleavable linking group that is cleaved at a desired pH, therebyreleasing the cationic lipid from the ligand inside the cell, or intothe desired compartment of the cell.

As a non-limiting example, a linker can include a cleavable linkinggroup that is cleavable by a particular enzyme. The type of cleavablelinking group incorporated into a linker can depend on the cell to betargeted. For example, liver targeting ligands can be linked to thecationic lipids through a linker that includes an ester group. Livercells are rich in esterases, and therefore the linker will be cleavedmore efficiently in liver cells than in cell types that are notesterase-rich. Other cell-types rich in esterases include cells of thelung, renal cortex, and testis.

As a non-limiting example, a linker can contain a peptide bond, whichcan be used when targeting cell types rich in peptidases, such as livercells and synoviocytes.

As a non-limiting example, suitability of a candidate cleavable linkinggroup can be evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. As a non-limiting example, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

In some embodiments, a linker comprises a redox cleavable linking group.As a non-limiting example, one class of cleavable linking groups areredox cleavable linking groups that are cleaved upon reduction oroxidation. A non-limiting example of reductively cleavable linking groupis a disulphide linking group (—S—S—). To determine if a candidatecleavable linking group is a suitable “reductively cleavable linkinggroup,” or for example is suitable for use with a particular iRNA moietyand particular targeting agent one can look to methods described herein.As a non-limiting example, a candidate can be evaluated by incubationwith dithiothreitol (DTT), or other reducing agent using reagents knowin the art, which mimic the rate of cleavage which would be observed ina cell, e.g., a target cell. The candidates can also be evaluated underconditions which are selected to mimic blood or serum conditions. As anon-limiting example, candidate compounds are cleaved by at most 10% inthe blood. As a non-limiting example, useful candidate compounds aredegraded at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

In some embodiments, a linker comprises a phosphate-based cleavablelinking groups are cleaved by agents that degrade or hydrolyze thephosphate group. An example of an agent that cleaves phosphate groups incells are enzymes such as phosphatases in cells. Examples ofphosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—,—O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—,—O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—,—S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—.Additional non-limiting examples are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. Anadditional non-limiting examples is —O—P(O)(OH)—O—.

In some embodiments, a linker comprises an acid cleavable linking groupsare linking groups that are cleaved under acidic conditions. As anon-limiting example, acid cleavable linking groups are cleaved in anacidic environment with a pH of about 6.5 or lower (e.g., about 6.0,5.5, 5.0, or lower), or by agents such as enzymes that can act as ageneral acid. In a cell, specific low pH organelles, such as endosomesand lysosomes can provide a cleaving environment for acid cleavablelinking groups. Examples of acid cleavable linking groups include butare not limited to hydrazones, esters, and esters of amino acids. Acidcleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O).In an additional non-limiting example, when the carbon attached to theoxygen of the ester (the alkoxy group) is an aryl group, substitutedalkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.

In some embodiments, a linker comprises an ester-based linking groups.As a non-limiting example, ester-based cleavable linking groups arecleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

In some embodiments, a linker comprises a peptide-based cleaving group.Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Asa non-limiting example, peptide-based cleavable groups do not includethe amide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynylene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins. Asa non-limiting example, a peptide based cleavage group can be limited tothe peptide bond (i.e., the amide bond) formed between amino acidsyielding peptides and proteins and does not include the entire amidefunctional group. As a non-limiting example, a peptide-based cleavablelinking groups can have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Any linker reported in the art can be used, including, as non-limitingexamples, those described in: U.S. Pat. App. No. 20150265708.

A non-limiting example of a method of conjugating a lipid and anoligonucleotide is presented in Example 1.

A non-limiting example of a linker is a C6 amino linker.

Target Components

In some embodiments, a provided composition further comprises atargeting component (targeting compound or moiety). A target componentcan be either conjugated or not conjugated to a lipid or a biologicallyactive agent. In some embodiments, a target component is conjugated to abiologically active agent. In some embodiments, a biologically activeagent is conjugated to both a lipid and a targeting component. Asdescribed in here, in some embodiments, a biologically active agent is aprovided oligonucleotide. Thus, in some embodiments, a providedoligonucleotide composition further comprises, besides a lipid andoligonucleotides, a target elements. Various targeting components can beused in accordance with the present disclosure, e.g., lipids,antibodies, peptides, carbohydrates, etc.

Target components can be incorporated into provided technologies throughmany types of methods in accordance with the present disclosure. In someembodiments, target components are physically mixed with providedoligonucleotides to form provided compositions. In some embodiments,target components are chemically conjugated with oligonucleotides.

In some embodiments, provided compositions comprise two or more targetcomponents. In some embodiments, provided oligonucleotides comprise twoor more conjugated target components. In some embodiments, the two ormore conjugated target components are the same. In some embodiments, thetwo or more conjugated target components are different. In someembodiments, provided oligonucleotides comprise no more than one targetcomponent. In some embodiments, oligonucleotides of a providedcomposition comprise different types of conjugated target components. Insome embodiments, oligonucleotides of a provided composition comprisethe same type of target components.

Target components can be conjugated to oligonucleotides optionallythrough linkers. Various types of linkers in the art can be utilized inaccordance of the present disclosure.

In some embodiments, a linker comprise a phosphate group, which can, forexample, be used for conjugating target components through chemistrysimilar to those employed in oligonucleotide synthesis. In someembodiments, a linker comprises an amide, ester, or ether group. In someembodiments, a linker has the structure of -L-. Target components can beconjugated through either the same or different linkers compared tolipids.

Target components, optionally through linkers, can be conjugated tooligonucleotides at various suitable locations. In some embodiments,target components are conjugated through the 5′-OH group. In someembodiments, target components are conjugated through the 3′-OH group.In some embodiments, target components are conjugated through one ormore sugar moieties. In some embodiments, target components areconjugated through one or more bases. In some embodiments, targetcomponents are incorporated through one or more internucleotidiclinkages. In some embodiments, an oligonucleotide may contain multipleconjugated target components which are independently conjugated throughits 5′-OH, 3′-OH, sugar moieties, base moieties and/or internucleotidiclinkages. Target components and lipids can be conjugated either at thesame, neighboring and/or separated locations. In some embodiments, atarget component is conjugated at one end of an oligonucleotide, and alipid is conjugated at the other end.

In some embodiments, the present disclosure provides the followingembodiments:

1. An oligonucleotide composition comprising a first plurality ofoligonucleotides which:

1) have a common base sequence; and

2) comprise one or more wing regions and a core region;

wherein:

each wing region comprises at least one modified sugar moiety; and

each core region comprises at least one un-modified sugar moiety.

2. An oligonucleotide composition comprising a first plurality ofoligonucleotides comprising one or more wing regions and a core region,wherein:

oligonucleotides of the first plurality have the same base sequence; and

each wing region independently comprises one or more modifiedinternucleotidic linkages and optionally one or more natural phosphatelinkages, and the core region independently comprises one or moremodified internucleotidic linkages; or

each wing region independently comprises one or more modified sugarmoieties, and the core region comprises one or more un-modified sugarmoieties.

3. An oligonucleotide composition, comprising a first plurality ofoligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence, for oligonucleotides of the particularoligonucleotide type.3a. An oligonucleotide composition, comprising a first plurality ofoligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

which composition is chirally controlled in that it is enriched,relative to a substantially racemic preparation of oligonucleotideshaving the same base sequence, for oligonucleotides of the particularoligonucleotide type,

wherein the oligonucleotides of the first plurality comprise one or morenatural phosphate linkages.

4. A composition of any one of the preceding embodiments, wherein theoligonucleotides comprise one or more wing regions and a core region,wherein:

oligonucleotides of the first plurality have the same base sequence;

each wing independently has a length of two or more bases, andindependently comprises one or more modified internucleotidic linkagesand optionally one or more natural phosphate linkages; and

the core region independently has a length of two or more bases andindependently comprises one or more modified internucleotidic linkages.

4a. A composition of any one of the preceding embodiments, wherein awing has the same or lower percentage of modified internucleotidiclinkages than the core.4b. A composition of any one of the preceding embodiments, wherein eachwing independently has the same or lower percentage of modifiedinternucleotidic linkages than the core.4c. A composition of any one of the preceding embodiments, wherein awing has a lower percentage of modified internucleotidic linkages thatthe core.4d. A composition of any one of the preceding embodiments, wherein awing has one or more natural phosphate linkages.4e. A composition of any one of the preceding embodiments, wherein eachwing independently has one or more natural phosphate linkages.4f. A composition of any one of the preceding embodiments, wherein has awing to the 5′ of the core has a modified internucleotidic linkage atits 5′-end.4g. A composition of any one of the preceding embodiments, wherein has awing to the 3′ of the core has a modified internucleotidic linkage atits 3′-end.4h. A composition of any one of the preceding embodiments, wherein awing comprises a modified internucleotidic linkage followed by one ormore natural phosphate linkages in the wing.4i. A composition of any one of the preceding embodiments, wherein awing to the 5′ of a core comprises a modified internucleotidic linkagefollowed by one or more natural phosphate linkages in the wing.4j. A composition of any one of the preceding embodiments, wherein, awing to the 5′ of a core comprises a modified internucleotidic linkagefollowed by two or more consecutive natural phosphate linkages in thewing.4k. A composition of any one of the preceding embodiments, wherein awing comprises a modified internucleotidic linkage preceded by one ormore natural phosphate linkages in the wing.4l. A composition of any one of the preceding embodiments, wherein awing to the 3′ of a core comprises a modified internucleotidic linkagepreceded by one or more natural phosphate linkages in the wing.4m. A composition of any one of the preceding embodiments, wherein awing to the 3′ of a core comprises a modified internucleotidic linkagepreceded by two or more consecutive natural phosphate linkages in thewing.4n. A composition of any one of the preceding embodiments, wherein awing is to the 5′-end of the core and comprises a natural phosphatelinkage between the two nucleosides at its 3′-end.4o. A composition of any one of the preceding embodiments, wherein awing is to the 3′-end of the core and comprises a natural phosphatelinkage between the two nucleosides at its 5′-end;5. A composition of embodiment 1, wherein the first plurality comprisesat least about 10% of the oligonucleotides in the composition.5a. A composition of embodiment 1, wherein the first plurality comprisesat least about 20% of the oligonucleotides in the composition.5b. A composition of embodiment 1, wherein the first plurality comprisesat least about 50% of the oligonucleotides in the composition.5c. A composition of embodiment 1, wherein the first plurality comprisesat least about 60% of the oligonucleotides in the composition.6. A composition of embodiment 1, wherein the first plurality comprisesat least about 70% of the oligonucleotides in the composition.7. A composition of embodiment 1, wherein the first plurality comprisesat least about 80% of the oligonucleotides in the composition.8. A composition of embodiment 1, wherein the first plurality comprisesat least about 85% of the oligonucleotides in the composition.9. A composition of embodiment 1, wherein the first plurality comprisesat least about 90% of the oligonucleotides in the composition.10. A composition of embodiment 1, wherein the first plurality comprisesat least about 95% of the oligonucleotides in the composition.11. A composition of any one of the preceding embodiments, comprising afirst plurality of oligonucleotides of a particular oligonucleotide typedefined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

12. A composition of any one of the preceding embodiments, wherein thecomposition comprises a first plurality of oligonucleotides of aparticular oligonucleotide type, wherein oligonucleotides of aparticular oligonucleotide type have a common pattern of basemodification and pattern of sugar modification.13. A composition of any one of the preceding embodiments, wherein thecomposition comprises a first plurality of oligonucleotides of aparticular oligonucleotide type, wherein oligonucleotides of aparticular oligonucleotide type are structurally identical.14. A composition of any one of the preceding embodiments, wherein thelevel of the first plurality of oligonucleotides is pre-determined.15. A composition of any one of the preceding embodiments, wherein afirst plurality of oligonucleotides comprise two wing regions and a coreregion.15a. A composition of any one of the preceding embodiments, wherein afirst plurality of oligonucleotides comprise no more than two wingregions and no more than one core region.16. A composition of any one of the preceding embodiments, wherein afirst plurality of oligonucleotides have a wing-core-wing structure.17. A composition of any one of the preceding embodiments, wherein afirst plurality of oligonucleotides are gapmers having a wing-core-wingstructure.18. A composition of any one of embodiments 1-13, wherein a firstplurality of oligonucleotides comprise no more than one wing region andno more than one core region.19. A composition of any one of embodiments 1-13, wherein a firstplurality of oligonucleotides are hemimers having a wing-core structure.20. A composition of any one of embodiments 1-13, wherein a firstplurality of oligonucleotides are hemimers having a core-wing structure.21. A composition of any one of the preceding embodiments, wherein awing comprises a chiral internucleotidic linkage.22. A composition of any one of the preceding embodiments, wherein awing comprises no more than 20% chiral internucleotidic linkages.23. A composition of any one of the preceding embodiments, wherein eachwing independently comprises a chiral internucleotidic linkage.24. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 20% chiral internucleotidiclinkages.25. A composition of any one of embodiments 1-17 and 19-22, wherein awing to the 5′-end of the core comprises a chiral internucleotidiclinkage at the 5′-end of the wing.26. A composition of any one of embodiments 1-16 and 18-22, wherein awing to the 3′-end of the core comprises a chiral internucleotidiclinkage at the 3′-end of the wing.27. A composition of any one of the preceding embodiments, wherein awing has only one chiral internucleotidic linkage, and each of the otherinternucleotidic linkages of the wing is a natural phosphate linkage

28. A composition of any one of the preceding embodiments, wherein thechiral internucleotidic linkage has the structure of formula I.29. A composition of any one of the preceding embodiments, wherein achiral internucleotidic linkage has the structure of formula I, andwherein X is S, and Y and Z are O.30. A composition of any one of the preceding embodiments, wherein achiral internucleotidic linkage is a phosphorothioate linkage.31. A composition of any one of the preceding embodiments, wherein achiral internucleotidic linkage is Sp.32. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is Sp.33. A composition of any one of embodiments 1-31, wherein a chiralinternucleotidic linkage is Rp.34. A composition of any one of embodiments 1-30, wherein each chiralinternucleotidic linkage is Rp.35. A composition of any one of embodiments 1-33, wherein a wingcomprises an Sp phosphorothioate linkage.36. A composition of any one of embodiments 1-33, wherein each wingindependently comprises an Sp phosphorothioate linkage.37. A composition of any one of embodiments 1-19, 21-33, and 35-36,wherein a wing is to the 5′-end of the core, and the wing has an Spphosphorothioate linkage.38. A composition of any one of embodiments 1-19, 21-33, and 35-37,wherein a wing is to the 5′-end of the core, and the wing has an Spphosphorothioate linkage at the 5′-end of the wing.39. A composition of any one of embodiments 1-19, 21-33, and 35-38,wherein a wing is to the 5′-end of the core, the wing has an Spphosphorothioate linkage at the 5′-end of the wing, and each of theother internucleotidic linkages of the wing is a natural phosphatelinkage

40. A composition of any one of embodiments 1-18, 20-33 and 35-36,wherein a wing is to the 3′-end of the core, and the wing has an Spphosphorothioate linkage at the 3′-end of the wing.41. A composition of any one of embodiments 1-18, 20-33, 35-36 and 40,wherein a wing is to the 3′-end of the core, and the wing has an Spphosphorothioate linkage at the 3′-end of the wing.42. A composition of any one of embodiments 1-18, 20-33, 35-36 and40-41, wherein one wing is to the 3′-end of the common core, the winghas an Sp phosphorothioate linkage at the 3′-end of the wing, and eachof the other internucleotidic linkages of the wing is a naturalphosphate linkage

43. A composition of any one of embodiments 1-31 and 33-42, wherein awing comprises an Rp phosphorothioate linkage.44. A composition of any one of embodiments 1-31 and 33-42, wherein eachwing independently comprises an Rp phosphorothioate linkage.45. A composition of any one of embodiments 1-19, 21-31 and 33-44,wherein a wing is to the 5′-end of the core, and the wing has an Rpphosphorothioate linkage.46. A composition of any one of embodiments 1-19, 21-31 and 33-45,wherein a wing is to the 5′-end of the core, and the wing has an Rpphosphorothioate linkage at the 5′-end of the wing.47. A composition of any one of embodiments 1-19, 21-31 and 33-46,wherein a wing is to the 5′-end of the core, the wing has an Rpphosphorothioate linkage at the 5′-end of the wing, and each of theother internucleotidic linkages of the wing is a natural phosphatelinkage

48. A composition of any one of embodiments 1-18, 20-31 and 33-44,wherein a wing is to the 3′-end of the core, and the wing has an Rpphosphorothioate.49. A composition of any one of embodiments 1-18, 20-31 and 33-44,wherein a wing is to the 3′-end of the core, and the wing has an Rpphosphorothioate linkage at the 3′-end of the wing.50. A composition of any one of embodiments 1-18, 20-31 and 33-44,wherein one wing is to the 3′-end of the common core, the wing has an Rpphosphorothioate linkage at the 3′-end of the wing, and each of theother internucleotidic linkages of the wing is a natural phosphatelinkage

51. A composition of any one of embodiments 1-30, wherein a wing is tothe 5′-end of a core, and its 5′-end internucleotidic linkage is achiral internucleotidic linkage.52. A composition of any one of embodiments 1-30, wherein a wing is tothe 5′-end of a core, and its 5′-end internucleotidic linkage is an Spchiral internucleotidic linkage.53. A composition of any one of embodiments 1-30, wherein a wing is tothe 5′-end of a core, and its 5′-end internucleotidic linkage is an Rpchiral internucleotidic linkage.54. A composition of any one of embodiments 1-30 and 51-53, wherein awing is to the 3′-end of a core, and its 3′-end internucleotidic linkageis a chiral internucleotidic linkage.55. A composition of any one of embodiments 1-30 and 51-53, wherein awing is to the 3′-end of a core, and its 3′-end internucleotidic linkageis an Sp chiral internucleotidic linkage.56. A composition of any one of embodiments 1-30 and 51-53, wherein awing is to the 3′-end of a core, and its 3′-end internucleotidic linkageis an Rp chiral internucleotidic linkage.57. A composition of any one of the preceding embodiments, wherein awing independently comprises a natural phosphate linkage

58. A composition of any one of the preceding embodiments, wherein awing independently comprises two or more natural phosphate linkages

59. A composition of any one of the preceding embodiments, wherein awing independently comprises two or more natural phosphate linkages, andall natural phosphate linkages within a wing are consecutive.60. A composition of any one of the preceding embodiments, wherein eachwing independently comprises a natural phosphate linkage

61. A composition of any one of the preceding embodiments, wherein eachwing independently comprises two or more natural phosphate linkages.61a. A composition of any one of the preceding embodiments, wherein eachwing independently comprises three or more natural phosphate linkages.61b. A composition of any one of the preceding embodiments, wherein eachwing independently comprises four or more natural phosphate linkages.61c. A composition of any one of the preceding embodiments, wherein eachwing independently comprises five or more natural phosphate linkages.62. A composition of any one of the preceding embodiments, wherein eachwing independently comprises two or more natural phosphate linkages, andall natural phosphate linkages within a wing are consecutive.62a. A composition of any one of the preceding embodiments, wherein eachwing independently comprises three or more natural phosphate linkages,and all natural phosphate linkages within a wing are consecutive.62b. A composition of any one of the preceding embodiments, wherein eachwing independently comprises four or more natural phosphate linkages,and all natural phosphate linkages within a wing are consecutive.62c. A composition of any one of the preceding embodiments, wherein eachwing independently comprises five or more natural phosphate linkages,and all natural phosphate linkages within a wing are consecutive.63. A composition of any one of the preceding embodiments, wherein atleast 5% of the internucleotidic linkages in a wing are naturalphosphate linkages.63a. A composition of any one of the preceding embodiments, wherein atleast 10% of the internucleotidic linkages in a wing are naturalphosphate linkages.63b. A composition of any one of the preceding embodiments, wherein atleast 20% of the internucleotidic linkages in a wing are naturalphosphate linkages.63c. A composition of any one of the preceding embodiments, wherein atleast 30% of the internucleotidic linkages in a wing are naturalphosphate linkages.63c. A composition of any one of the preceding embodiments, wherein atleast 40% of the internucleotidic linkages in a wing are naturalphosphate linkages.63e. A composition of any one of the preceding embodiments, wherein atleast 50% of the internucleotidic linkages in a wing are naturalphosphate linkages.63f. A composition of any one of the preceding embodiments, wherein atleast 60% of the internucleotidic linkages in a wing are naturalphosphate linkages.63g. A composition of any one of the preceding embodiments, wherein atleast 70% of the internucleotidic linkages in a wing are naturalphosphate linkages.63h. A composition of any one of the preceding embodiments, wherein atleast 80% of the internucleotidic linkages in a wing are naturalphosphate linkages.63i. A composition of any one of the preceding embodiments, wherein atleast 90% of the internucleotidic linkages in a wing are naturalphosphate linkages.63j. A composition of any one of the preceding embodiments, wherein atleast 95% of the internucleotidic linkages in a wing are naturalphosphate linkages.64. A composition of any one of the preceding embodiments, wherein atleast 5% of the internucleotidic linkages in each wing are independentlynatural phosphate linkages.64a. A composition of any one of the preceding embodiments, wherein atleast 10% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64b. A composition of any one of the preceding embodiments, wherein atleast 20% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64c. A composition of any one of the preceding embodiments, wherein atleast 30% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64c. A composition of any one of the preceding embodiments, wherein atleast 40% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64e. A composition of any one of the preceding embodiments, wherein atleast 50% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64f. A composition of any one of the preceding embodiments, wherein atleast 60% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64g. A composition of any one of the preceding embodiments, wherein atleast 70% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64h. A composition of any one of the preceding embodiments, wherein atleast 80% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64i. A composition of any one of the preceding embodiments, wherein atleast 90% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.64j. A composition of any one of the preceding embodiments, wherein atleast 95% of the internucleotidic linkages in each wing areindependently natural phosphate linkages.65. A composition of any one of the preceding embodiments, wherein awing comprises no more than 15 modified phosphate linkages.65a. A composition of any one of the preceding embodiments, wherein awing comprises no more than 10 modified phosphate linkages.65b. A composition of any one of the preceding embodiments, wherein awing comprises no more than 9 modified phosphate linkages.65c. A composition of any one of the preceding embodiments, wherein awing comprises no more than 8 modified phosphate linkages.65d. A composition of any one of the preceding embodiments, wherein awing comprises no more than 7 modified phosphate linkages.65e. A composition of any one of the preceding embodiments, wherein awing comprises no more than 6 modified phosphate linkages.65f. A composition of any one of the preceding embodiments, wherein awing comprises no more than 5 modified phosphate linkages.65g. A composition of any one of the preceding embodiments, wherein awing comprises no more than 4 modified phosphate linkages.65h. A composition of any one of the preceding embodiments, wherein awing comprises no more than 3 modified phosphate linkages.65i. A composition of any one of the preceding embodiments, wherein awing comprises no more than 2 modified phosphate linkages.65j. A composition of any one of the preceding embodiments, wherein awing comprises no more than 1 modified phosphate linkage.66. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 15 modified phosphatelinkages.66a. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 10 modified phosphatelinkages.66b. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 9 modified phosphate linkages.66c. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 8 modified phosphate linkages.66d. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 7 modified phosphate linkages.66e. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 6 modified phosphate linkages.66f. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 5 modified phosphate linkages.66g. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 4 modified phosphate linkages.66h. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 3 modified phosphate linkages.66i. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 2 modified phosphate linkages.66j. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 1 modified phosphate linkage.67. A composition of any one of the preceding embodiments, wherein awing comprises less than 100% modified phosphate linkages.67a. A composition of any one of the preceding embodiments, wherein awing comprises no more than 95% modified phosphate linkages.67b. A composition of any one of the preceding embodiments, wherein awing comprises no more than 90% modified phosphate linkages.67c. A composition of any one of the preceding embodiments, wherein awing comprises no more than 80% modified phosphate linkages.67d. A composition of any one of the preceding embodiments, wherein awing comprises no more than 70% modified phosphate linkages.67e. A composition of any one of the preceding embodiments, wherein awing comprises no more than 60% modified phosphate linkages.67f. A composition of any one of the preceding embodiments, wherein awing comprises no more than 50% modified phosphate linkages.67g. A composition of any one of the preceding embodiments, wherein awing comprises no more than 40% modified phosphate linkages.67h. A composition of any one of the preceding embodiments, wherein awing comprises no more than 30% modified phosphate linkages.67i. A composition of any one of the preceding embodiments, wherein awing comprises no more than 20% modified phosphate linkages.67k. A composition of any one of the preceding embodiments, wherein awing comprises no more than 10% modified phosphate linkage.68. A composition of any one of the preceding embodiments, wherein eachwing independently comprises less than 100% modified phosphate linkages.68a. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 95% modified phosphatelinkages.68b. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 90% modified phosphatelinkages.68c. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 80% modified phosphatelinkages.68d. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 70% modified phosphatelinkages.68e. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 60% modified phosphatelinkages.68f. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 50% modified phosphatelinkages.68g. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 40% modified phosphatelinkages.68h. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 30% modified phosphatelinkages.68i. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 20% modified phosphatelinkages.68j. A composition of any one of the preceding embodiments, wherein eachwing independently comprises no more than 10% modified phosphatelinkage.69. A composition of any one of the preceding embodiments, wherein aninternucleotidic linkage of a wing region is independently selected froma natural phosphate linkage and a modified phosphate linkage having thestructure of formula I.70. A composition of any one of the preceding embodiments, wherein aninternucleotidic linkage of a wing region is independently selected froma natural phosphate linkage and a phosphorothioate linkage.71. A composition of any one of embodiments 1-20, wherein eachinternucleotidic linkage within a wing is a natural phosphate linkage.71a. A composition of any one of embodiments 1-20, wherein eachinternucleotidic linkage within each wing is a natural phosphatelinkage.72. A composition of any one of embodiments 1-20, wherein eachinternucleotidic linkage within a wing is a chiral internucleotidiclinkage.72a. A composition of any one of embodiments 1-20, wherein eachinternucleotidic linkage within each wing is a chiral internucleotidiclinkage.73. A composition of any one of the preceding embodiments, wherein awing has a length of three or more bases.73a. A composition of any one of the preceding embodiments, wherein onewing has a length of four or more bases.73b. A composition of any one of the preceding embodiments, wherein onewing has a length of five or more bases.73c. A composition of any one of the preceding embodiments, wherein onewing has a length of six or more bases.73d. A composition of any one of the preceding embodiments, wherein onewing has a length of seven or more bases.73e. A composition of any one of the preceding embodiments, wherein onewing has a length of eight or more bases.73f. A composition of any one of the preceding embodiments, wherein onewing has a length of nine or more bases.73g. A composition of any one of the preceding embodiments, wherein onewing has a length of ten or more bases.74. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of three or more bases.74a. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of four or more bases.74b. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of five or more bases.74c. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of six or more bases.74d. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of seven or more bases.74e. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of eight or more bases.74f. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of nine or more bases.74g. A composition of any one of the preceding embodiments, wherein eachwing independently has a length of ten or more bases.75. A composition of any one of embodiments 1-72, wherein a wing has alength of three bases.76. A composition of any one of embodiments 1-72, wherein a wing has alength of four bases.77. A composition of any one of embodiments 1-72, wherein a wing has alength of five bases.78. A composition of any one of embodiments 1-72, wherein a wing has alength of six bases.79. A composition of any one of embodiments 1-72, wherein a wing has alength of seven bases.80. A composition of any one of embodiments 1-72, wherein a wing has alength of eight bases.81. A composition of any one of embodiments 1-72, wherein a wing has alength of nine bases.82. A composition of any one of embodiments 1-72, wherein a wing has alength of ten bases.83. A composition of any one of embodiments 1-72, wherein a wing has alength of 11 bases.84. A composition of any one of embodiments 1-72, wherein a wing has alength of 12 bases.85. A composition of any one of embodiments 1-72, wherein a wing has alength of 13 bases.86. A composition of any one of embodiments 1-72, wherein a wing has alength of 14 bases.87. A composition of any one of embodiments 1-72, wherein a wing has alength of 15 bases.88. A composition of any one of embodiments 1-72, wherein each wing hasthe same length.89. A composition of any one of the preceding embodiments, wherein awing is defined by sugar modifications relative to a core.90. A composition of any one of the preceding embodiments, wherein eachwing independently comprises a modified sugar moiety.91. A composition of any one of the preceding embodiments, wherein eachwing sugar moiety is independently a modified sugar moiety.92. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a high-affinity sugar modification.93. A composition of any one of the preceding embodiments, wherein amodified sugar moiety has a 2′-modification.94. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a bicyclic sugar modification.95. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a bicyclic sugar modification having a-L- or —O-L- bridge connecting two ring carbon atoms.96. A composition of any one of the preceding embodiments, wherein amodified sugar moiety comprises a bicyclic sugar modification having a4′-CH(CH₃)—O-2′ bridge.97. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-OR¹.98. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-OR¹, wherein R¹ is optionally substituted C₁₋₆ alkyl.99. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-MOE.100. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein a 2′-modification is2′-OMe.101. A composition of any one of embodiments 1-96, wherein a modifiedsugar moiety comprises a 2′-modification, wherein the 2′-modification isS-cEt.102. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein the 2′-modification isFANA.103. A composition of any one of embodiments 1-93, wherein a modifiedsugar moiety comprises a 2′-modification, wherein the 2′-modification isFRNA.104. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety has a 5′-modification.105. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety is R-5′-Me-DNA.106. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety is S-5′-Me-DNA.107. A composition of any one of embodiments 1-92, wherein a modifiedsugar moiety is FHNA.108. A composition of any one of the preceding embodiments, wherein eachwing sugar moiety is modified.109. A composition of any one of the preceding embodiments, wherein allmodified wing sugar moieties within a wing have the same modification.110. A composition of any one of the preceding embodiments, wherein allmodified wing sugar moieties have the same modification.111. A composition of any one of embodiments 1-108, wherein at least onemodified wing sugar moiety is different than another modified wing sugarmoiety.112. A composition of any one of the preceding embodiments, wherein awing comprises a modified base.113. A composition of any one of the preceding embodiments, wherein awing comprises a 2S-dT.114. A composition of any one of the preceding embodiments, wherein thecore region has a length of five or more bases.115. A composition of any one of the preceding embodiments, wherein thecore region has a length of six or more bases.116. A composition of any one of the preceding embodiments, wherein thecore region has a length of seven or more bases.117. A composition of any one of the preceding embodiments, wherein thecore region has a length of eight or more bases.118. A composition of any one of the preceding embodiments, wherein thecore region has a length of nine or more bases.119. A composition of any one of the preceding embodiments, wherein thecore region has a length of ten or more bases.120. A composition of any one of the preceding embodiments, wherein thecore region has a length of 11 or more bases.121. A composition of any one of the preceding embodiments, wherein thecore region has a length of 12 or more bases.122. A composition of any one of the preceding embodiments, wherein thecore region has a length of 13 or more bases.123. A composition of any one of the preceding embodiments, wherein thecore region has a length of 14 or more bases.124. A composition of any one of the preceding embodiments, wherein thecore region has a length of 15 or more bases.125. A composition of any one of 1-113, wherein the core region has alength of five bases.126. A composition of any one of 1-113, wherein the core region has alength of six bases.127. A composition of any one of 1-113, wherein the core region has alength of seven bases.128. A composition of any one of 1-113, wherein the core region has alength of eight bases.129. A composition of any one of 1-113, wherein the core region has alength of nine bases.130. A composition of any one of 1-113, wherein the core region has alength of ten bases.131. A composition of any one of 1-113, wherein the core region has alength of 11 bases.132. A composition of any one of 1-113, wherein the core region has alength of 12 bases.133. A composition of any one of 1-113, wherein the core region has alength of 13 bases.134. A composition of any one of 1-113, wherein the core region has alength of 14 bases.135. A composition of any one of 1-113, wherein the core region has alength of 15 bases.136. A composition of any one of the preceding embodiments, wherein thecore region does not have any 2′-modification.153. A composition of any one of the preceding embodiments, wherein eachcore sugar moiety is not modified.138. A composition of any one of the preceding embodiments, wherein eachsugar moiety of the core region is the natural DNA sugar moiety.139. A composition of any one of the preceding embodiments, wherein thecore region comprises a chiral internucleotidic linkage.140. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage.141. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage having the structure of formula I.142. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage having the structure of formula I, and wherein X is S, and Y andZ are 0.143. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage of the core region is a chiral internucleotidiclinkage having the structure of formula I, and wherein one -L-R¹ is not—H.144. A composition of any one of embodiments 1-142, wherein eachinternucleotidic linkage of the core region is a phosphorothioatelinkage.145. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral center comprises(Sp)m(Rp)n, wherein m is 1-50, and n is 1-10.146. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral center comprises(Sp)m(Rp)n, wherein m is 1-50, n is 1-10, and m>n.147. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral center comprises(Sp)m(Rp)n, wherein m is 2, 3, 4, 5, 6, 7 or 8, and n is 1.148. A composition of any one of embodiments 1-144, wherein the coreregion has a pattern of backbone chiral centers comprising (Rp)n(Sp)m,wherein m is 1-50 and n is 1-10.149. A composition of any one of embodiments 1-144 and 148, wherein thecore region has a pattern of backbone chiral centers comprising Rp(Sp)m,wherein m is 2, 3, 4, 5, 6, 7 or 8.150. A composition of any one of embodiments 1-144 and 148-149, whereinthe core region has a pattern of backbone chiral centers comprisingRp(Sp)₂.151. A composition of any one of embodiments 1-144, wherein the coreregion has a pattern of backbone chiral centers comprising(Np)t(Rp)n(Sp)m, wherein t is 1-10, n is 1-10, m is 1-50, and each Np isindependent Rp or Sp.152. A composition of any one of embodiments 1-144 and 151, wherein thecore region has a pattern of backbone chiral centers comprising(Sp)t(Rp)n(Sp)m, wherein t is 1-10, n is 1-10, m is 1-50.153. A composition of any one of embodiments 1-144 and 151-152, whereinn is 1.154. A composition of any one of embodiments 1-144 and 151-153, whereint is 2, 3, 4, 5, 6, 7 or 8.155. A composition of any one of embodiments 1-144 and 151-154, whereinm is 2, 3, 4, 5, 6, 7 or 8.156. A composition of any one of embodiments 1-144 and 151-155, whereinat least one of t and m is greater than 5.157. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral centers comprisingSpSpRpSpSp.158. A composition of any one of the preceding embodiments, wherein 50%or more of the chiral internucleotidic linkages in the core region haveSp configuration.159. A composition of any one of the preceding embodiments, wherein 60%or more of the chiral internucleotidic linkages in the core region haveSp configuration.160. A composition of any one of the preceding embodiments, wherein 70%or more of the chiral internucleotidic linkages in the core region haveSp configuration.161. A composition of any one of the preceding embodiments, wherein 80%or more of the chiral internucleotidic linkages in the core region haveSp configuration.162. A composition of any one of the preceding embodiments, wherein 90%or more of the chiral internucleotidic linkages in the core region haveSp configuration.163. A composition of any one of the preceding embodiments, wherein eachinternucleotidic linkage in the core region is chiral, the core regionhas only one Rp, and each of the other internucleotidic linkages in thecore region is Sp.164. A composition of any one of the preceding embodiments, wherein eachbase moiety in the core is not modified.165. A composition of any one of embodiments 1-163, wherein the coreregion comprises a modified base.166. A composition of any one of embodiments 1-163, wherein the coreregion comprises a modified base, wherein a modified base is substitutedA, T, C or G.167. A composition of any one of embodiments 1-164, wherein each basemoiety in the core region is independently selected from A, T, C and G.168. A composition of any one of embodiments 1-163, wherein the coreregion is a DNA sequence whose phosphate linkages are independentlyreplaced with phosphorothioate linkages.169. A composition of any one of the preceding embodiments, wherein theoligonucleotides are single stranded.170. A composition of any one of the preceding embodiments, wherein theoligonucleotides are antisense oligonucleotide, antagomir, microRNA,pre-microRNs, antimir, supermir, ribozyme, U1 adaptor, RNA activator,RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide,aptamer or adjuvant.171. A composition of any one of the preceding embodiments, wherein theoligonucleotides are antisense oligonucleotides.172. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 10 bases.173. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 11 bases.174. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 12 bases.175. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 13 bases.176. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 14 bases.177. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 15 bases.178. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 16 bases.179. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 17 bases.180. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 18 bases.181. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 19 bases.182. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 20 bases.183. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 21 bases.184. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 22 bases.185. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 23 bases.186. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 24 bases.187. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of greater than 25 bases.188. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 200 bases.189. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 150 bases.190. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 100 bases.191. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 50 bases.192. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 40 bases.193. A composition of any one of the preceding embodiments, wherein theoligonucleotides have a length of less than about 30 bases.194. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 10 bases.195. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 11 bases.196. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 12 bases.197. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 13 bases.198. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 14 bases.199. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 15 bases.200. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 16 bases.201. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 17 bases.202. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 18 bases.203. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 19 bases.204. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 20 bases.205. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 21 bases.206. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 22 bases.207. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 23 bases.207a. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 24 bases.207b. A composition of any one of embodiments 1-171, wherein theoligonucleotides have a length of 25 bases.208. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality have a pattern of backbone chiralcenter comprises (Sp)m(Rp)n, wherein m is 1-50, and n is 1-10.208a. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality a pattern of backbone chiralcenter comprises (Sp)m(Rp)n, wherein m is 1-50, n is 1-10, and m>n.208b. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality a pattern of backbone chiralcenter comprises (Sp)m(Rp)n, wherein m is 2, 3, 4, 5, 6, 7 or 8, and nis 1.208c. A composition of any one of embodiments 1-207, whereinoligonucleotides of a first plurality a pattern of backbone chiralcenters comprising (Rp)n(Sp)m, wherein m is 1-50 and n is 1-10.208d. A composition of any one of embodiments 1-207 and 208d, whereinoligonucleotides of a first plurality a pattern of backbone chiralcenters comprising Rp(Sp)m, wherein m is 2, 3, 4, 5, 6, 7 or 8.208e. A composition of any one of embodiments 1-207 and 208d-208e,wherein oligonucleotides of a first plurality a pattern of backbonechiral centers comprising Rp(Sp)₂.208f. A composition of any one of embodiments 1-207, whereinoligonucleotides of a first plurality a pattern of backbone chiralcenters comprising (Np)t(Rp)n(Sp)m, wherein t is 1-10, n is 1-10, m is1-50, and each Np is independent Rp or Sp.208g. A composition of any one of embodiments 1-207 and 208g, whereinoligonucleotides of a first plurality a pattern of backbone chiralcenters comprising (Sp)t(Rp)n(Sp)m, wherein t is 1-10, n is 1-10, m is1-50.208h. A composition of any one of embodiments 1-207 and 208g-208f,wherein n is 1.208i. A composition of any one of embodiments 1-207 and 208g-208g,wherein t is 2, 3, 4, 5, 6, 7 or 8.208j. A composition of any one of embodiments 1-207 and 208g-208h,wherein m is 2, 3, 4, 5, 6, 7 or 8.208k. A composition of any one of embodiments 1-207 and 208g-208i,wherein at least one of t and m is greater than 5.208l. A composition of any one of the preceding embodiments, wherein thecore region has a pattern of backbone chiral centers comprisingSpSpRpSpSp.209. A composition of any one of the preceding embodiments, wherein 50%or more of the chiral internucleotidic linkages in oligonucleotides of afirst plurality have Sp configuration.209a. A composition of any one of the preceding embodiments, wherein 60%or more of the chiral internucleotidic linkages in oligonucleotides of afirst plurality have Sp configuration.209b. A composition of any one of the preceding embodiments, wherein 70%or more of the chiral internucleotidic linkages in oligonucleotides of afirst plurality have Sp configuration.209c. A composition of any one of the preceding embodiments, wherein 80%or more of the chiral internucleotidic linkages in oligonucleotides of afirst plurality have Sp configuration.209d. A composition of any one of the preceding embodiments, wherein 90%or more of the chiral internucleotidic linkages in oligonucleotides of afirst plurality have Sp configuration.209e. A composition of any one of the preceding embodiments, whereineach internucleotidic linkage in oligonucleotides of a first pluralityis chiral, the core region has only one Rp, and each of the otherinternucleotidic linkages in oligonucleotides of a first plurality isSp.210. A composition of any one of the preceding embodiments, wherein theoligonucleotide type is not (Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO: 60) or(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp,Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC(5R—(SSR)3-5R) (SEQ ID NO: 61), wherein in the underlined nucleotide are2′-O-MOE modified.211. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from: (Sp, Sp, Rp,Sp, Sp, Rp, Sp, Sp, Rp, Sp,Sp)-d[5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (SEQ ID NO: 60) or(Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp,Rp)-Gs5mCs5mCsTs5mCsAsGsTs5mCsTsGs5mCsTsTs5mCsGs5mCsAs5mCs5mC(5R—(SSR)₃₋₅R) (SEQ ID NO: 61), wherein in the underlined nucleotide are2′-O-MOE modified.212. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from:

ONT-106 (Rp)-uucuAGAccuGuuuuGcuudT

dT PCSK9 sense (SEQ ID NO: 62) ONT-107 (Sp)-uucuAGAccuGuuuuGcuudT

dT PCSK9 sense (SEQ ID NO: 63) ONT-108 (Rp)-AAGcAAAAcAGGUCuAGAAdT

dT PCSK9  (SEQ ID NO: 64) antisense ONT-109 (Sp)-AAGcAAAAcAGGUCuAGAAdT

dT PCSK9  (SEQ ID NO: 65) antisense ONT-110 (Rp, Rp)-a

AGcAAAAcAGGUCuAGAA PCSK9 dT

dT (SEQ ID NO: 66) antisense ONT-111 (Sp, Rp)-a

GcAAAAcAGGUCuAGAAd PCSK9 T

dT (SEQ ID NO: 67) antisense ONT-112 (Sp, Sp)-a

GcAAAAcAGGUCuAGAAd PCSK9 T

dT (SEQ ID NO: 68) antisense ONT-113 (Rp, Sp)-a

GcAAAAcAGGUCuAGAAd PCSK9 T

dT (SEQ ID NO: 69) antisensewherein lower case letters represent 2′OMe RNA residues; capital lettersrepresent 2′OH RNA residues; and bolded and “s” indicates aphosphorothioate moiety; and

PCSK9 (1) (All (Sp))-ususcsusAsGsAscscsusGsususususGscsususdTsdT (SEQ ID NO: 70) PCSK9 (2)(All (Rp))-ususcsusAsGsAscscsusGsususu susGscsususdTsdT (SEQ ID NO: 71)PCSK9 (3) (All (Sp))-usucuAsGsAsccuGsuuuuGscuusd TsdT (SEQ ID NO: 72)PCSK9 (4) (All (Rp))-usucuAsGsAsccuGsuuuuGscuusd TsdT (SEQ ID NO: 73)PCSK9 (5) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-ususcsusAsGsAscscsusGsususususGscsus usdTsdT (SEQ ID NO: 74)PCSK9 (6) (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-ususcsusAsGsAscscsusGsususususGscsususdT sdT (SEQ ID NO: 75)wherein lower case letters represent 2′-OMe RNA residues; capitalletters represent RNA residues; d=2′-deoxy residues; and “s” indicates aphosphorothioate moiety; and

PCSK9 (7) (All (Rp))-AsAsGscsAsAsAsAscsAsGsGsUsCsusAsGsAsAsdTsdT (SEQ ID NO: 76) PCSK9 (8)(All (Sp))-AsAsGscsAsAsAsAscsAsGsGsUs CsusAsGsAsAsdTsdT (SEQ ID NO: 77)PCSK9 (9) (All (Rp))-AsAGcAAAAcsAsGsGsUsCsusAsGsAsAsdTsdT (SEQ ID NO: 78) PCSK9 (10)(All (Sp))-AsAGcAAAAcsAsGsGsUsCsusAsG sAsAsdTsdT (SEQ ID NO: 79)PCSK9 (11) (All (Rp))-AAsGscsAsAsAsAscAGGUCuAGAA dTsdT (SEQ ID NO: 80)PCSK9 (12) (All (Sp))-AAsGscsAsAsAsAscAGGUCuAGAA dTsdT (SEQ ID NO: 81)PCSK9 (13) (All (Rp))-AsAsGscAsAsAsAscAsGsGsUsCsuAsGsAsAsdTsdT (SEQ ID NO: 82) PCSK9 (14)(All (Sp))-AsAsGscAsAsAsAscAsGsGsUsCs uAsGsAsAsdTsdT (SEQ ID NO: 83)PCSK9 (15) (All (Rp))-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT (SEQ ID NO: 84) PCSK9 (16)(All (Sp))-AsAGcAAAsAscAsGsGsUsCsusAs GsAsAsdTsdT (SEQ ID NO: 85)PCSK9 (17) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp)-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT (SEQ ID NO: 86) PCSK9 (18)(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp)-AsAGcAAAsAscAsGsGsUsCsusAsGsAsAsdTsdT (SEQ ID NO: 87)wherein lower case letters represent 2′-OMe RNA residues; capitalletters represent RNA residues; d=2′-deoxy residues; “s” indicates aphosphorothioate moiety; and

PCSK9 (19) (All (Rp))-UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT (SEQ ID NO: 88) PCSK9 (20)(All (Sp))-UfsusCfsusAfsgsAfscsCfsus GfsusUfsusUfsgsCfsusUfsdTsdT(SEQ ID NO: 89) PCSK9 (21) (All (Rp))-UfsuCfsuAfsgAfscCfsuGfsuUfsuUfsgCfsuUfsdTsdT (SEQ ID NO: 90) PCSK9 (22)(All (Sp))-UfsuCfsuAfsgAfscCfsuGfsuU fsuUfsgCfsuUfsdTsdT (SEQ ID NO: 91)PCSK9 (23) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-UfsusCfsusAfsgsAfscsCfsusGfsusUfsusUfsgsCfsusUfsdTsdT (SEQ ID NO: 92) PCSK9 (24)(Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp,Rp)-UfsusCfsusAfsgsAfscsCfsusGf susUfsusUfsgsCfsusUfsdTsdT(SEQ ID NO: 93)wherein lower case letters represent 2′-OMe RNA residues; capitalletters represent 2′-F RNA residues; d=2′-deoxy residues; and “s”indicates a phosphorothioate moiety; and

PCSK9 (25) (All (Rp))-asAfsgsCfsasAfsasAfscsAfsgsGfsusCfsusAfsgsAfsasdTsdT (SEQ ID NO: 94) PCSK9 (26)(All (Sp))-asAfsgsCfsasAfsasAfscsAfsg sGfsusCfsusAfsgsAfsasdTsdT(SEQ ID NO: 95) PCSK9 (27) (All (Rp))-asAfgCfaAfaAfcsAfsgsGfsusCfsusAfsgsAfsasdTsdT (SEQ ID NO: 96) PCSK9 (28)(All (Sp))-asAfgCfaAfaAfcsAfsgsGfsusC fsusAfsgsAfsasdTsdT(SEQ ID NO: 97) PCSK9 (29) (All (Rp))-asAfsgCfsaAfsaAfscAfsgGfsuCfsuAfsgAfsadTsdT (SEQ ID NO: 98) PCSK9 (30)(All (Sp))-asAfsgCfsaAfsaAfscAfsgGfsu CfsuAfsgAfsadTsdT (SEQ ID NO: 99)PCSK9 (31) (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp,Sp, Rp, Sp, Rp, Sp)-asAfgCfaAfasAfscA fsgsGfsusCfsusAfsgsAfsasdTsdT(SEQ ID NO: 100) PCSK9 (32) (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp,Rp, Sp, Rp, Sp, Rp)-asAfgCfaAfasAf scAfsgsGfsusCfsusAfsgsAfsasdTsdT(SEQ ID NO: 101)213. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from:d[A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C_(S)A_(R)C] (SEQ ID NO: 102),d[C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C_(S)C_(S)C] (SEQ ID NO: 103),d[C_(S)C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C] (SEQ ID NO: 104) andd[C_(S)C_(S)C_(S)C_(S)C_(S)C_(R)C_(R)C_(S)C_(S)C] (SEQ ID NO: 105),wherein R is Rp phosphorothioate linkage, and S is Sp phosphorothioatelinkage. 214. A composition of any one of the preceding embodiments,wherein the oligonucleotide is not an oligonucleotide selected from:GGA_(R)T_(S)G_(R)T_(S)T_(R) ^(m)C_(S)TCGA (SEQ ID NO: 106),GGA_(R)T_(R)G_(S)T_(S)T_(R) ^(m)C_(R)TCGA (SEQ ID NO: 107),GGA_(S)T_(S)G_(R)T_(S)T_(S) ^(m)C_(S)TCGA (SEQ ID NO: 108), wherein R isRp phosphorothioate linkage, S is Sp phosphorothioate linkage, all otherlinkages are PO, and each ^(m)C is a 5-methyl cytosine modifiednucleoside. 215. A composition of any one of the preceding embodiments,wherein the oligonucleotide is not an oligonucleotide selected from:T_(k)T_(k) ^(m)C_(k)AGT^(m)CATGA^(m)CT_(k)T^(m)C_(k) ^(m)C_(k) (SEQ IDNO: 109), wherein each nucleoside followed by a subscript ‘k’ indicatesa (S)-cEt modification, R is Rp phosphorothioate linkage, S is Spphosphorothioate linkage, each ^(m)C is a 5-methyl cytosine modifiednucleoside, and all internucleoside linkages are phosphorothioates (PS)with stereochemistry patterns selected from RSSSRSRRRS, RSSSSSSSSS,SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS,SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR,RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS,RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS, RRRSSRRSRS,SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR and SSSSSSSSSS.216. A composition of any one of the preceding embodiments, wherein theoligonucleotide is not an oligonucleotide selected from: T_(k)T_(k)^(m)C_(k) AGT^(m)CATGA^(m)CTT_(k) ^(m)C_(k) ^(m)C_(k) (SEQ ID NO: 110),wherein each nucleoside followed by a subscript ‘k’ indicates a (S)-cEtmodification, R is Rp phosphorothioate linkage, S is Sp phosphorothioatelinkage, each ^(m)C is a 5-methyl cytosine modified nucleoside and allinternucleoside linkages in the underlined core are phosphorothioates(PS) with stereochemistry patterns selected from: RSSSRSRRRS,RSSSSSSSSS, SRRSRSSSSR, SRSRSSRSRSSRSS, RRRSRSSRSR, RRSSSRSRSR,SRSSSRSSSS, SSRRSSRSRS, SSSSRRSS, RRRSSRRRSR, RRRRSSSRS, SRRSRRRRRR,RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR,RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, RSRSRSSRSS,RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRSR, RRRRRRRRRR andSSSSSSSSSS.217. A composition of embodiment 215 or 216, wherein eachphosphorothioate moiety of each nucleotide comprising (S)-cEtmodification is stereorandom.218. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise two or more naturalphosphate linkages.218a. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise three or more naturalphosphate linkages.218b. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise four or more naturalphosphate linkages.218c. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise five or more naturalphosphate linkages.218d. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise six or more naturalphosphate linkages.218e. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise seven or more naturalphosphate linkages.218f. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise eight or more naturalphosphate linkages.218g. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise nine or more naturalphosphate linkages.218h. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise ten or more naturalphosphate linkages.219. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise two or more modifiedphosphate linkages.219a. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise three or more modifiedphosphate linkages.219b. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise four or more modifiedphosphate linkages.219c. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise five or more modifiedphosphate linkages.219d. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise six or more modifiedphosphate linkages.219e. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise seven or more modifiedphosphate linkages.219f. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise eight or more modifiedphosphate linkages.219g. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise nine or more modifiedphosphate linkages.219i. A composition of any one of the preceding embodiments, whereinoligonucleotides of a first plurality comprise ten or more modifiedphosphate linkages.220. A composition of any one of embodiments 219-219i, wherein eachmodified phosphate linkage is a phosphorothioate linkage.221. A composition of any one of the preceding embodiments, wherein afirst plurality of oligonucleotides comprises a modified base, wherein amodified base is substituted A, T, C, U or G.222. A composition of any one of the preceding embodiments, wherein eachbase moiety in an oligonucleotide of the first plurality is optionallysubstituted A, T, C, U, or G.223. A composition of any one of the preceding embodiments, wherein eachbase moiety in an oligonucleotide of the first plurality isindependently selected from A, T, C, U, 5-MeC and G.224. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is formed with greater than 90:10diastereomeric selectivity.225. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is formed with greater than 95:5diastereomeric selectivity.226. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is formed with greater than 96:4diastereomeric selectivity.227. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is formed with greater than 97:3diastereomeric selectivity.228. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is formed with greater than 98:2diastereomeric selectivity.229. A composition of any one of the preceding embodiments, wherein eachchiral internucleotidic linkage is formed with greater than 98:2diastereomeric selectivity.230. A composition of any one of embodiments 224-229, wherein thediastereomeric selectivity for forming a chiral internucleotidic linkageis measured by forming a dimeric oligonucleotide comprising the chiralinternucleotidic linkage and the nucleosides to both sides of the chiralinternucleotidic linkage under the same or comparable reactionconditions.231. A composition of any one of the preceding embodiments, whichcomposition displays reduced toxicity as compared with a referencecomposition comprising a plurality of oligonucleotides, each of whichalso has the common base sequence but which differs structurally fromthe oligonucleotides of the first plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

231a. A composition of any one of the preceding embodiments, whichcomposition displays reduced immune activation as compared with areference composition comprising a plurality of oligonucleotides, eachof which also has the common base sequence but which differsstructurally from the oligonucleotides of the first plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

231b. A composition of any one of the preceding embodiments, whichcomposition displays reduced complement activation as compared with areference composition comprising a plurality of oligonucleotides, eachof which also has the common base sequence but which differsstructurally from the oligonucleotides of the first plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

231c. A composition of any one of the preceding embodiments, whichcomposition displays reduced injection site inflammation as comparedwith a reference composition comprising a plurality of oligonucleotides,each of which also has the common base sequence but which differsstructurally from the oligonucleotides of the first plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

232. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detects aprotein whose level changes upon complement activation.233. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of one or morecomplete-activation related product.234. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of one or morecomplete-activation related product selected from the group consistingof C3a, Bb, C4a, C5a, C5b, C6, C7, C8 and C9.234a. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of C3a.234b. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of C4a.234c. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of C5a.234d. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of Bb.235. A composition of any one of the preceding embodiments, wherein thereduced complement activation is observed in monkey serum.236. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 5%.236a. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 10%.236b. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 20%.236c. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 30%.236d. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 40%.236e. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 50%.236f. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 60%.236g. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 70%.236h. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 80%.236i. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 90%.236j. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 95%.236k. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 96%.236l. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 97%.236m. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 98%.236n. A composition of any one of the preceding embodiments, whereincomplement activation is reduced by 99%.237. A composition of any one of the preceding embodiments, whereincomplement activation is measured at a time point less than 60 minutesafter the composition is administered.237a. A composition of any one of the preceding embodiments, whereincomplement activation is measured at a time point within 0-40 minutesafter the composition is administered.237b. A composition of any one of the preceding embodiments, whereincomplement activation is measured at multiple time points.238. A composition of any one of the preceding embodiments, whereincomplement activation is measured at a time point as measured by C₃alevels at a time point within the range of 5-60 minutes after thecomposition is added to monkey serum.239. A composition of any one of the preceding embodiments, whichcomposition displays altered protein binding as compared with areference composition comprising a plurality of oligonucleotides, eachof which also has the common base sequence but which differsstructurally from the oligonucleotides of the first plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

240. A composition of any one of the preceding embodiments, wherein thecomposition displays altered binding as compared with a referencecomposition to one or more proteins selected from serum proteins,heparin sulfate-binding proteins, and intracellular proteins.241. A composition of any one of the preceding embodiments, wherein thecomposition displays altered binding as compared with a referencecomposition to one or more proteins selected from Albumin, ComplementFactor H, Factor IX, ApoE, Thrombin, Factor VIIIa, Heparin Cofactor II,alpha-2 macroglobulin, Fibroblast Growth Factor 1, Fibroblast GrowthFactor 2, Hepatocyte Growth Factor/Scatter Factor, Vascular EndothelialGrowth Factor, High-Mobility Group Protein B1, Cyclophilin B, IL-8(CXCL8), Platelet Factor 4 (CXCL4), Stromal Cell-Derived Factor-1(CXCL12), Monocyte Chemoattractant Protein-1 (CCL2), Fibroblast GrowthFactor Receptor 1, Neuropilin-1, Receptor for Advanced Glycation EndProducts, Receptor Protein Tyrosine Phosphatase Sigma, Slit-2, ROBO1,Thrombin, Antithrombin, Protein C inhibitor, Amyloid precursor protein1, Thrombospondin-1, Annexin A2, PDGF BB, PC4/Sub1, RNF163/ZNF9, Ku70,Ku80, TCP -alpha, TCP -beta, TCP -epsilon, TCP -gamma, TCP1-Theta,TCP1-delta, HSP90-AA1, HSP90-AB, HSP70-5/GRP78, HSPA1L, HSC70, ACTB,TBBB2C, Vimentin, CArG Binding Factor, DHX30, EIF2S2, EIF4H, GRSF1,hnRNP D1L, hnRNPA1, hnRNPA2, hnRNPH1, hnRNPK, hnRNPQ, hnRNPU, hnRNPUL,ILF2, ILF3, KHSRP, La/SSB, NCL, NPM1, P54nrb, PSF, PSPC1, RHA, YBX1,ACLY, VARS, ANXA2, NDKA, Thymidylate Kinase, JKBP1 delta 6, SHMT2,LRPPRC, NARS, ATAD3A, KCTD12, CD4, GP120, aMb2 (Mac-1), VDAC-1, Ago2 PAZdomain, RAGE, AIM2, DHX36, DHX9, DDX41, IFI16, RIG-I, MDA5, LRRFIP1,DLM-1/ZBP1, TREX1, Laminin, and Fibronectin.242. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins.242a. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 10%.242b. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 20%.242c. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 30%.242d. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 40%.242e. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 50%.242f. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 60%.242g. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 70%.242h. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 80%.242i. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 90%.242j. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 95%.242k. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 96%.242l. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 97%.242m. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 98%.242n. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to one or more proteins by more than 99%.243. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to Factor H.243a. A composition of any one of the preceding embodiments, wherein thecomposition displays decreased binding as compared with a referencecomposition to a heparin sulfate binding protein.244. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition to one or more proteins.245. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition to albumin.245a. A composition of any one of the preceding embodiments, wherein theincreased protein binding is observed in a BSA binding assay, whereinthe first plurality of oligonucleotides have increased BSA binding thatthe reference plurality.246. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 5% or more.246a. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 10% or more.246b. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 20% or more.246c. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 30% or more.246d. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 40% or more.246e. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 50% or more.246f. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 60% or more.246g. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 70% or more.246h. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 80% or more.246i. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 90% or more.246j. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 100% or more.246k. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 2 folds or more.246l. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 5 folds or more.246m. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 10 folds or more.246n. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 50 folds or more.246o. A composition of any one of the preceding embodiments, wherein thecomposition displays increased binding as compared with a referencecomposition by 100 folds or more.246p. A composition of any one of the preceding embodiments, whereinprotein binding is measured in vitro.247. A composition of any one of the preceding embodiments, which thecomposition displays improved oligonucleotide delivery as compared witha reference composition comprising a plurality of oligonucleotides, eachof which also has the common base sequence but which differsstructurally from the oligonucleotides of the first plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

247a. A composition of any one of the preceding embodiments, wherein thecomposition displays improved systemic delivery.247b. A composition of any one of the preceding embodiments, wherein thecomposition displays improved cytoplasmatic delivery.247c. A composition of any one of the preceding embodiments, wherein thecomposition displays improved delivery to a target.247d. A composition of any one of the preceding embodiments, wherein thecomposition displays improved delivery to a population of cells.247e. A composition of any one of the preceding embodiments, wherein thecomposition displays improved delivery to a tissue.247f. A composition of any one of the preceding embodiments, wherein thecomposition displays improved delivery to an organ.248. A composition of any one of the preceding embodiments, furthercomprising a pharmaceutically acceptable carrier.248a. A composition of any one of the preceding embodiments, whereinoligonucleotides of the first plurality are for treatment of a diseaseassociated with complement.248b. A composition of any one of the preceding embodiments, whereinoligonucleotides of the first plurality are for treatment ofneuroinflammation, neurodegeneration, muscular inflammation,demyelination, vasculitis or nephritis.248c. A composition of any one of the preceding embodiments, whereinoligonucleotides of the first plurality are for treatment of lupusnephritis.248d. A composition of any one of the preceding embodiments, whereinoligonucleotides of the first plurality target C1, C1a, C1r, C1s, C1q,MASP-1, MASP-2, C3, C3-convertase, C3a, C3b, C3aR, C4b, C5, C5a, C5aR,Factor B, Factor D, Thrombin, Plasmin, Kallikrein, or FactorXIIa.248e. A composition of any one of the preceding embodiments, whereinoligonucleotides of the first plurality target C5.248f. A composition of any one of the preceding embodiments exceptembodiment 248e, wherein oligonucleotides of the first plurality targetfactor B.248g. A composition of any one of the preceding embodiments, comprisingone or more lipids.248h. A composition of any one of the preceding embodiments, wherein atleast one lipid is conjugated to the oligonucleotides of thecomposition.248i. A composition of any one of the preceding embodiments, wherein atleast one lipid is covalently conjugated to the oligonucleotides of thecomposition.248j. A composition of any one of the preceding embodiments, comprisingone or more targeting components.248k. A composition of any one of the preceding embodiments, wherein atleast one target component is conjugated to the oligonucleotides of thecomposition.248l. A composition of any one of the preceding embodiments, wherein atleast one target component is covalently conjugated to theoligonucleotides of the composition.249. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is characterized by reduced toxicityrelative to a reference oligonucleotide composition of the same commonnucleotide sequence.

249a. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is chirally controlled and that ischaracterized by reduced toxicity relative to a referenceoligonucleotide composition of the same common nucleotide sequence.

249b. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises: administering anoligonucleotide composition of any one of the preceding embodiments.249c. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises: administering anoligonucleotide composition in which each oligonucleotide in theplurality comprises one or more modified sugar moieties and thecomposition is characterized by reduced toxicity relative to a referenceoligonucleotide composition of the same common nucleotide sequence butlacking at least one of the one or more modified sugar moieties.249d. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises: administering anoligonucleotide composition in which each oligonucleotide in theplurality includes one or more natural phosphate linkages and one ormore modified phosphate linkages; wherein the oligonucleotidecomposition is characterized by reduced toxicity when tested in at leastone assay that is observed with an otherwise comparable referencecomposition whose oligonucleotides do not comprise natural phosphatelinkages.250. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises: administering anoligonucleotide composition in which each oligonucleotide in theplurality comprises one or more modified sugar moieties and thecomposition is characterized by reduced toxicity relative to a referenceoligonucleotide composition of the same common nucleotide sequence butlacking at least one of the one or more modified sugar moieties.251. A method comprising steps of administering to a subject anoligonucleotide composition comprising a first plurality ofoligonucleotides each of which has a common base sequence and comprisesa modified sugar moiety, wherein the oligonucleotide composition ischaracterized by reduced toxicity when tested in at least one assay thatis observed with an otherwise comparable reference composition thatcomprises a reference plurality of oligonucleotides which have the samecommon base sequence but have no modified sugar moieties.252. A method comprising steps of administering to a subject anoligonucleotide composition comprising a first plurality ofoligonucleotides each of which has a common base sequence and comprisesone or more natural phosphate linkages and one or more modifiedphosphate linkages, wherein the oligonucleotide composition ischaracterized by reduced toxicity when tested in at least one assay thatis observed with an otherwise comparable reference composition thatcomprises a reference plurality of oligonucleotides which have the samecommon base sequence but have no natural phosphate linkages.253. A method comprising steps of administering a chirally controlledoligonucleotide composition to a subject, wherein the chirallycontrolled oligonucleotide composition is characterized by reducedtoxicity when tested in at least one assay that is observed with anotherwise comparable reference composition that includes a differentchirally controlled oligonucleotide composition, or a stereorandomoligonucleotide composition, comprising oligonucleotides having the samebase sequence.254. A method of administering an oligonucleotide composition comprisinga first plurality of oligonucleotides having a common nucleotidesequence, comprising:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is characterized by reduced toxicityrelative to a reference oligonucleotide composition comprising areference plurality of oligonucleotides of the same common nucleotidesequence.

254a. A method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, comprising:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is characterized by reducedcomplement activation relative to a reference oligonucleotidecomposition comprising a reference plurality of oligonucleotides of thesame common nucleotide sequence.

254b. A method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, comprising:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is characterized by altered proteinbinding relative to a reference oligonucleotide composition comprising areference plurality of oligonucleotides of the same common nucleotidesequence.

254c. The method of embodiment 254b, wherein the altered protein bindingcomprises improved binding to albumin.254d. A method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, comprising:

administering an oligonucleotide composition comprising the firstplurality of oligonucleotides that is characterized by reduced injectionsite inflammation relative to a reference oligonucleotide compositioncomprising a reference plurality of oligonucleotides of the same commonnucleotide sequence.

254e. A method of any one of the preceding embodiments, wherein thereduced toxicity is or comprises reduced complement activation.254f. A method of any one of the preceding embodiments, wherein thereduced toxicity is assessed in a complement activation assay.254g. A method of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detects aprotein whose level changes upon complement activation.254h. A method of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of one or morecomplete-activation related product.254i. A method of any one of the preceding embodiments, wherein thereduced complement activation is observed in an assay that detectspresence, absolute level and or relative levels of one or morecomplete-activation related product selected from the group consistingof C₃a, Bb or combinations thereof.254j. A method of any one of the preceding embodiments, wherein thereduced complement activation is observed in monkey serum.255. A method of any one of the preceding embodiments, whereinoligonucleotides of the administered oligonucleotide compositions arefor treatment of a disease associated with complement.255a. A method of any one of the preceding embodiments, whereinoligonucleotides of the administered oligonucleotide compositions arefor treatment of neuroinflammation, neurodegeneration, muscularinflammation, demyelination, vasculitis or nephritis.255b. A method of any one of the preceding embodiments, whereinoligonucleotides of the administered oligonucleotide compositions arefor treatment of lupus nephritis.255c. A method of any one of the preceding embodiments, whereinoligonucleotides of the administered oligonucleotide compositions targetC1, C1a, C1r, C1s, C1q, MASP-1, MASP-2, C3, C3-convertase, C3a, C3b,C3aR, C4b, C5, C5a, CSaR, Factor B, Factor D, Thrombin, Plasmin,Kallikrein, or FactorXIIa.255d. A method of any one of the preceding embodiments, whereinoligonucleotides of the administered oligonucleotide compositions targetC5.255e. A method of any one of the preceding embodiments except embodiment255d, wherein oligonucleotides of the administered oligonucleotidecompositions target factor B.256. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 5%.256a. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 10%.256b. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 20%.256c. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 30%.256d. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 40%.256e. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 50%.256f. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 60%.256g. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 70%.256h. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 80%.256i. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 90%.256j. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 95%.256k. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 96%.256l. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 97%.256m. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 98%.256n. A method of any one of the preceding embodiments, whereincomplement activation is reduced by at least 99%.256o. A method of any one of the preceding embodiments, whereincomplement activation is reduced to a level comparable to a negativecontrol.256p. A method of any one of the preceding embodiments, whereincomplement activation is reduced to a level comparable to water.257. A method of any one of the preceding embodiments, whereincomplement activation is measured at a time point no more than 60minutes after the composition is added to monkey serum.257a. A method of any one of the preceding embodiments, whereincomplement activation is measured at a time point 0-40 minutes after thecomposition is added to monkey serum.258. A method of any one of the preceding embodiments, whereincomplement activation is measured at a time point as measured by C3alevels at a time point within the range of 5-60 minutes after thecomposition is added to monkey serum.259. A method of any one of the preceding embodiments, wherein thecomposition of the first plurality of oligonucleotides displays reducedinjection site inflammation as compared with a reference composition.260. A method, comprising administering a composition comprising a firstplurality of oligonucleotides, which composition displays reducedinjection site inflammation as compared with a reference compositioncomprising a plurality of oligonucleotides, each of which also has thecommon base sequence but which differs structurally from theoligonucleotides of the first plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

260a. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises: administering anoligonucleotide comprising a first plurality of oligonucleotides that ischaracterized by reduced injection site inflammation relative to areference oligonucleotide composition of the same common nucleotidesequence.261. A method of any one of the preceding embodiments, wherein thecomposition of the first plurality of oligonucleotides displays alteredprotein binding.262. A method, comprising administering a composition comprising a firstplurality of oligonucleotides, which composition displays alteredprotein binding as compared with a reference composition comprising aplurality of oligonucleotides, each of which also has the common basesequence but which differs structurally from the oligonucleotides of thefirst plurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

262a. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises: administering anoligonucleotide composition comprising a first plurality ofoligonucleotides that is characterized by altered protein bindingrelative to a reference oligonucleotide composition of the same commonnucleotide sequence.263. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysaltered binding as compared with a reference composition to one or moreproteins selected from serum proteins, heparin sulfate-binding proteins,and intracellular proteins.264. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysaltered binding as compared with a reference composition to one or moreproteins selected from Albumin, Complement Factor H, Factor IX, ApoE,Thrombin, Factor VIIIa, Heparin Cofactor II, alpha-2 macroglobulin,Fibroblast Growth Factor 1, Fibroblast Growth Factor 2, HepatocyteGrowth Factor/Scatter Factor, Vascular Endothelial Growth Factor,High-Mobility Group Protein B1, Cyclophilin B, IL-8 (CXCL8), PlateletFactor 4 (CXCL4), Stromal Cell-Derived Factor-1 (CXCL12), MonocyteChemoattractant Protein-1 (CCL2), Fibroblast Growth Factor Receptor 1,Neuropilin-1, Receptor for Advanced Glycation End Products, ReceptorProtein Tyrosine Phosphatase Sigma, Slit-2, ROBO1, Thrombin,Antithrombin, Protein C inhibitor, Amyloid precursor protein 1,Thrombospondin-1, Annexin A2, PDGF BB, PC4/Sub1, RNF163/ZNF9, Ku70,Ku80, TCP1-alpha, TCP1-beta, TCP1-epsilon, TCP1-gamma, TCP1-Theta,TCP1-delta, HSP90-AA1, HSP90-AB, HSP70-5/GRP78, HSPA1L, HSC70, ACTB,TBBB2C, Vimentin, CArG Binding Factor, DHX30, EIF2S2, EIF4H, GRSF1,hnRNP D1L, hnRNPA1, hnRNPA2, hnRNPH1, hnRNPK, hnRNPQ, hnRNPU, hnRNPUL,ILF2, ILF3, KHSRP, La/SSB, NCL, NPM1, P54nrb, PSF, PSPC1, RHA, YBX1,ACLY, VARS, ANXA2, NDKA, Thymidylate Kinase, JKBP1 delta 6, SHMT2,LRPPRC, NARS, ATAD3A, KCTD12, CD4, GP120, aMb2 (Mac-1), VDAC-1, Ago2 PAZdomain, RAGE, AIM2, DHX36, DHX9, DDX41, IFI16, RIG-I, MDA5, LRRFIP1,DLM-1/ZBP1, TREX1, Laminin, and Fibronectin.265. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins.265a. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 10%.265b. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 20%.265c. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 30%.265d. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 40%.265e. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 50%.265f. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 60%.265g. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 70%.265h. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 80%.265i. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 90%.265j. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 95%.265k. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 96%.265l. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 97%.265m. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 98%.265n. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to one ormore proteins by more than 99%.266. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to Factor H.266. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysdecreased binding as compared with a reference composition to a heparinsulfate binding protein.267. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition to one ormore proteins.268. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition to albumin.268a. A method of any one of the preceding embodiments, wherein theincreased protein binding is observed in a BSA binding assay, whereinthe first plurality of oligonucleotides have increased BSA binding thatthe reference plurality.269. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 5% ormore.269a. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 10% ormore.269b. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 20% ormore.269c. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 30% ormore.269d. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 40% ormore.269e. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 50% ormore.269f. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 60% ormore.269g. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 70% ormore.269h. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 80% ormore.269i. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 90% ormore.269j. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 100% ormore.269k. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 2 folds ormore.269l. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 5 folds ormore.269m. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 10 foldsor more.269n. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 50 foldsor more.269o. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysincreased binding as compared with a reference composition by 100 foldsor more.270. A method of any one of the preceding embodiments, wherein proteinbinding is measured in vitro.271. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysimproved delivery.272. A method, comprising administering a composition comprising a firstplurality of oligonucleotides, which composition displays improveddelivery as compared with a reference composition comprising a pluralityof oligonucleotides, each of which also has the common base sequence butwhich differs structurally from the oligonucleotides of the firstplurality in that:

individual oligonucleotides within the reference plurality differ fromone another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have astructure different from a structure represented by the plurality ofoligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do notcomprise a wing region and a core region.

273. In a method of administering an oligonucleotide compositioncomprising a first plurality of oligonucleotides having a commonnucleotide sequence, the improvement that comprises: administering anoligonucleotide comprising a first plurality of oligonucleotides that ischaracterized by improved delivery relative to a referenceoligonucleotide composition of the same common nucleotide sequence.274. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysimproved systemic delivery.274a. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysimproved cytoplasmatic delivery.275. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysimproved delivery to a target.276. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysimproved delivery to a population of cells.276a. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysimproved delivery to a tissue.276b. A method of any one of the preceding embodiments, wherein thecomposition comprising the first plurality of oligonucleotides displaysimproved delivery to an organ.277. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more modifiedinternucleotidic linkages than oligonucleotides of the referencecomposition.277a. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more phosphorothioatelinkages than oligonucleotides of the reference composition.277b. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more phosphorothioatelinkages than oligonucleotides of the reference composition at the5′-end.277c. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more phosphorothioatelinkages than oligonucleotides of the reference composition at the3′-end.277d. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more phosphorothioatelinkages in a wing region than oligonucleotides of the referencecomposition.277e. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more phosphorothioatelinkages in each wing region than oligonucleotides of the referencecomposition.278. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more Sp chiralinternucleotidic linkages than oligonucleotides of the referencecomposition.278a. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more Spphosphorothioate linkages than oligonucleotides of the referencecomposition.278b. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more Spphosphorothioate linkages than oligonucleotides of the referencecomposition at the 5′-end.278c. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more Spphosphorothioate linkages than oligonucleotides of the referencecomposition at the 3′-end.278d. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more Spphosphorothioate linkages in a wing region than oligonucleotides of thereference composition.278e. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more Spphosphorothioate linkages in each wing region than oligonucleotides ofthe reference composition.279. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more modified basesthan oligonucleotides of the reference composition.279a. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more methylated basesthan oligonucleotides of the reference composition.279b. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more methylated basesthan oligonucleotides of the reference composition at the 5′-end.279c. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more methylated basesthan oligonucleotides of the reference composition at the 3′-end.279d. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more methylated basesthan in a wing region than oligonucleotides of the referencecomposition.279e. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise more methylated basesthan in each wing region than oligonucleotides of the referencecomposition.280. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise fewer 2′-MOEmodifications than oligonucleotides of the reference composition.280a. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise fewer 2′-MOEmodifications than oligonucleotides of the reference composition.281. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise fewer 2′-MOEmodifications than oligonucleotides of the reference composition at the5′-end.282. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise fewer 2′-MOEmodifications than oligonucleotides of the reference composition at the3′-end.283. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise fewer 2′-MOEmodifications than in a wing region than oligonucleotides of thereference composition.284. A method of any one of the preceding embodiments, whereinoligonucleotides of the first plurality comprise fewer 2′-MOEmodifications than in each wing region than oligonucleotides of thereference composition.285. A composition or method of any one of the preceding embodiments,wherein individual oligonucleotides within the reference pluralitydiffer from one another in stereochemical structure.286. A composition or method of any one of the preceding embodiments,wherein at least some oligonucleotides within the reference pluralityhave a structure different from a structure represented by the pluralityof oligonucleotides of the composition.287. A composition or method of any one of the preceding embodiments,wherein at least some oligonucleotides within the reference plurality donot comprise a wing region and a core region.288. A method of any one of the preceding embodiments, wherein acomposition of a first plurality of oligonucleotides is a composition ofany one of the preceding embodiments.289. A method of decreasing level of a target nucleic acid in a cell,tissue, and/or organism without significant complement activation bycontacting with a composition of any one of the preceding embodiments.290. A method of directing RNase H cleavage of a target nucleic acidwithout significant complement activation by contacting the targetnucleic acid with a composition of any one of the preceding embodiments.291. A method of any one of the preceding embodiments, wherein themethod is performed under conditions and for a time sufficient to reducelevel of the target nucleic acid sequence.292. A method of identifying and/or characterizing an oligonucleotidecomposition, the method comprising steps of:

providing at least one composition of any one of the precedingembodiments;

assessing complement activation relative to a reference composition.

293. A method of identifying and/or characterizing an oligonucleotidecomposition, the method comprising steps of:

providing at least one composition of any one of the precedingembodiments;

assessing injection site inflammation relative to a referencecomposition.

294. A method of identifying and/or characterizing an oligonucleotidecomposition, the method comprising steps of:

providing at least one composition of any one of the precedingembodiments;

assessing protein binding relative to a reference composition.

295. A method of identifying and/or characterizing an oligonucleotidecomposition, the method comprising steps of:

providing at least one composition of any one of the precedingembodiments;

assessing delivery relative to a reference composition.

296. A method or composition of any one of the preceding embodiments,wherein the reference composition is a substantially racemic preparationof oligonucleotides that share the base sequence.297. A method or composition of any one of the preceding embodiments,wherein the reference composition is a chirally controlledoligonucleotide composition of another oligonucleotide type.298. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise morephosphorothioate linkages.299. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise onlyphosphorothioate linkages.300. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewermodified sugar moieties.301. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewermodified sugar moieties, wherein the modification is 2′-OR¹.302. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise moremodified sugar moieties.303. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise moremodified sugar moieties, wherein the modification is 2′-OR¹.304. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewerphosphorothioate linkages.305. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition have a wing, andcomprise fewer phosphorothioate linkages at the wing.306. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewer Spphosphorothioate linkages.307. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition have a wing, andcomprise fewer Sp phosphorothioate linkages at the wing.308. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise more Rpphosphorothioate linkages.309. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition have a wing, andcomprise more Rp phosphorothioate linkages at the wing.310. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewermethylated bases.311. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise more2′-MOE modifications.312. A method or composition of any one of the preceding embodiments,whereinoligonucleotides of the reference composition share the same:1) base sequence;2) pattern of backbone linkages; and3) pattern of backbone phosphorus modifications.313. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewernatural phosphate linkages.314. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewernatural phosphate linkages at the 5′- and/or 3′-end.315. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise fewernatural phosphate linkages in a region corresponding to a wing ofoligonucleotides of the first plurality.316. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the first plurality comprise naturalphosphate linkages in a wing, and oligonucleotides of the referencecomposition comprise fewer natural phosphate linkages at thecorresponding wing region.317. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the first plurality comprise naturalphosphate linkages in a wing, and oligonucleotides of the referencecomposition comprises modified internucleotidic linkages at one or moresuch natural phosphate linkage locations in a wing.318. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the first plurality comprise naturalphosphate linkages in a wing, and oligonucleotides of the referencecomposition comprises phosphorothioate linkages at one or more suchnatural phosphate linkage locations in a wing.319. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise nonatural phosphate linkages.320. A method or composition of any one of the preceding embodiments,wherein oligonucleotides of the reference composition comprise nowing-core-wing structure.321. A method for manufacturing an oligonucleotide composition directedto a selected target sequence, the method comprising steps of:

manufacturing an oligonucleotide composition comprising a firstplurality of oligonucleotides of any one of the preceding embodiments,each of which has a base sequence complementary to the target sequence.

322. A method of embodiment 321, further comprising providing apharmaceutically acceptable carrier.

EXEMPLIFICATION

The foregoing has been a description of certain non-limiting embodimentsof the disclosure. Accordingly, it is to be understood that theembodiments of the disclosure herein described are merely illustrativeof the application of the principles of the disclosure. Reference hereinto details of the illustrated embodiments is not intended to limit thescope of the claims.

Methods for preparing provided oligonucleotides and oligonucleotidecompositions are widely known in the art, including but not limited tothose described in WO/2010/064146, WO/2011/005761, WO/2013/012758,WO/2014/010250, US2013/0178612, WO/2014/012081 and WO/2015/107425, themethods and reagents of each of which is incorporated herein byreference. Applicant describes herein example methods for makingprovided oligonucleotides.

Example 1. Example Preparation of Linkers

In some embodiments, an SP linker was prepared following the schemebelow:

Example 2. Example Methods for Preparing Oligonucleotides andCompositions

Abbreviation

AMA: conc. NH₃—40% MeNH₂ in H₂O (1:1, v/v)CMIMT: N-cyanomethylimidazolium triflateDBU: 1,8-diazabicyclo[5.4.0]undec-7-eneDCA: dichloroacetic acidDCM: dichloromethane, CH₂Cl₂DMTr: 4,4′-dimethoxytritylDVB: divinylbenzeneHCP: highly cross-linked polystyrene (contains 50% DVB, non-swellingpolystyrene)

Melm: N-methylimidazole

MQ: water obtained from “Milli-Q Reference”PhlMT: N-phenylimidazolium triflatePOS: 3-phenyl-1,2,4-dithiazolin-5-onePS200: primer support 200, commercially available from GE HealthcarePS5G: primer support 5G, commercially available from GE HealthcareTBAF: tetrabutylammonium fluorideTBHP: tert-butylhydroperoxideTEAA: triethylammonium aceate

Solid support: Various types of solid support (varied nucleosidesloading) were tested. In some embodiments, HCP>PS5G≈PS200≥CPG. In someembodiments, a solid support is HCP. In some embodiments, a solidsupport is PS5G. In some embodiments, a solid support is PS200. In someembodiments, a solid support is CPG. For nucleosides loading, variousrange (30-300 μmol/g) were tested. In some embodiments, 70-80 μmol/gloading performed than others. In some embodiments, nucleoside loadingis 70-80 μmol/g. CPG was purchased from various suppliers (GlenReseach,LinkTechnologies, ChemGenes, PrimeSynthesis, and 3-Prime).

Various linkers were tested and can be used. In some embodiments, duringpreparation of chirally controlled oligonucleotide compositions by usingDPSE-type chemistry, SP-linker was used.

Various activators were prepared and/or purchased, and evaluated. Insome embodiments, for DPSE-type chemistry, CMIMT was used.

Example Analytical conditions:

RP-UPLC-MS System: Waters, Aquity UPLC I-Class, Xevo G2-Tof Column:Waters, BEH C18, 1.7 μm, 2.1×150 mm

Temp. & Flow rate: 55° C., 0.3 mL/min

Buffer: A: 0.1M TEAA; B: MeCN Gradient: % B: 1-30%/30 min AEX-HPLC

System: Waters, Alliance e2695

Column: Thermo, DNAPac PA-200, 4×250 mm

Temp. & Flow rate: 50° C., 1 mL/min

Buffer: A: 20 mM NaOH; B: A+1M NaClO₄ Gradient: % B: 10-50%/30 min

Example procedure for the synthesis of chrial-oligos (1 μmol scale):

Automated solid-phase synthesis of chiral-oligos was performed accordingto example cycles shown herein. After the synthesis cycles, the resinwas treated with 0.1M TBAF in MeCN (1 mL) for 2 h (30 min usuallyenough) at room temperature, washed with MeCN, dried, and add AMA (1 mL)for 30 min at 45° C. The mixture was cooled to room temperature and theresin was removed by membrane filtration. The filtrate was concentratedunder reduced pressure to about 1 mL. The residue was diluted with 1 mLof H₂O and analyzed by AEX-HPLC and RP-UPLC-MS (example conditions:refer to the analytical conditions).

waiting step operation reagents and solvent volume time 1 detritylation3% DCA in toluene 10 mL 65 s 2 coupling 0.15M monomer in ^(i)PrCN + 0.5mL 5 min 0.5M CMIMT in MeCN 3 capping 20% Ac₂O, 30% 2,6- 1.2 mL 60 slutidine in MeCN + 20% MeIm in MeCN 4 oxidation or 1.1M TBHP in DCM- 1.0mL 300 s sulfurization decane or 0.1M POS in MeCN

As described, in some embodiments, TBAF treatment can provide betterresults, for example, less desulfurization. In some embodiments, SPlinker provided better yields and/or purity through, without theintention to be limited by theory, better stability during chiralauxiliary removal as described. In some embodiments, fluoro-containingreagents such as HF—NR₃ (e.g., HF-TEA (triethylamine)), provided betteryields and/or purity when succinyl linker was used by, without theintention to be limited by theory, less cleavage during chiral auxiliaryremoval. In some embodiments, after synthesis, the resin was treatedwith 1M TEA-HF in DMF-H₂O (3:1, v/v; 1 mL) for 2 h at 50° C. PS5Gsupport was washed with MeCN, H₂O, and add AMA (conc. NH₃—40% MeNH₂(1:1, v/v)) (1 mL) for 45 min at 50° C. The mixture was cooled to roomtemperature and the resin was removed by membrane filtration (washedwith H₂O for 2 mL). The filtrate was concentrated under reduced pressureuntil it becomes about 1 mL. The residue was diluted with 1 mL of H₂Oand analyzed by AEX-HPLC and RP-UPLC-MS (conditions: refer to theanalytical conditions section).

Example procedure for the purification of chrial-oligos (1 μmol scale):in some embodiments, crude oligos were purified by AEX-MPLC according tothe following example conditions:

System: AKTA Purifier-10 Column: TOHSOH, DNA STAT, 4.6×100 mm

Temp. & Flow rate: 60° C., 0.5 mL/min

Buffer: A: 20 mM Tris-HCl (pH 9.0)+20% MeCN, B: A+1.5M NaCl Gradient: %B: 20-70%/25CV (2%/CV)

All fractions were analyzed by analytical AEX-HPLC, and fractionscontaining chiral oligo more than 80% purity were corrected and desaltedby Sep-Pak Plus tC18 (WAT036800) using example conditions below:

Conditioning Sep-Pak Plus with 15 mL of MeCN.Rinse cartridge with 15 mL of 50% MeCN/MQ.Equilibrate cartridge with 30 mL of MQ.Load sample, and wash with 40 mL of MQ.Elute chiral oligos with 10 mL of 50% MeCN/MQ.

Eluted sample were evapolated under reduced pressure to remove MeCN, andlyophilized. The product were dissolved in MQ (1 mL), filtered by 0.2 μmmesh syringe filter, and analyzed. After yield calculation by UVabsorbance, the preparation was lyophilized again.

Example methods, conditions and reagents were described in, e.g., JP2002-33436, WO2005/092909, WO2010/064146, WO2012/039448, WO2011/108682,WO2014/010250, WO2014/010780, WO2014/012081, etc., and may be useful forpreparing provided oligonucleotides and/or compositions.

Provided compositions, among other things, demonstrated improvedproperties including improved stability and activities. For example,provided oligonucleotide compositions provided increased cleavage rates,increased selectivity, enhanced cleavage pattern, etc. The examples heredemonstrated that provided compositions also have low toxicities andimproved protein binding profiles.

Example 3. Measurement of Cynomolgus Monkey Serum Complement ActivationIn Vitro

Effects of oligonucleotides on complement activation were measured invitro in Cynomolgus monkey serum. The third complement component, C3, iscentral to the classical, alternative and lectin pathways of complementactivation. During complement activation, C3 is proteolytically cleavedresulting in release of the anaphylatoxic peptide C3a. Upon activationof the alternative pathway, Factor B is cleaved by complement Factor Dyielding the noncatalytic chain Ba and the catalytic subunit Bb. Theactive subunit Bb is a serine protease that associates with C3b to formthe alternative pathway C3 convertase.

Serum from 3 individual Cynomolgus male monkeys was pooled and the poolwas used. The time course of C3a and Bb complement activation wasmeasured by incubating oligonucleotides at 37° C. at a finalconcentration of 330 ug/ml in Cynomolgus monkey serum (1:10 ratio, V/V)and taking aliquots at the indicated time points. Specifically, 9.24 μLof 10 mg/mL stock of oligonucleotide were added to 270.76 μL of pooledserum, incubated at 37° C. At the indicated time points, 20 μL aliquotswere taken out and the reaction was immediately terminated by additionof 2.2 μL of 18 mg/mL EDTA (Sigma-Aldrich).

For the dose response curves six 1/3 serial dilutions (10× concentrated)of oligonucleotides in water were prepared starting from 1 mg/mL. 2 μLof diluted oligonucleotide solutions were then added to 18 μL ofCynomolgus monkey serum and incubated at 37° C. After 40 min thereaction was immediately terminated by addition of 2.2 μL of 18 mg/mLEDTA (Sigma-Aldrich). C3a and Bb were measured using MicroVue C3a Plusand Bb Plus Enzyme Immunoassays from (Quidel, San Diego, Calif.) at1:3000 (C₃a) and 1:40 (Bb) dilution.

Example results were presented in FIGS. 1-5. In some embodiments,provided compositions have altered levels of complement activationcompared the reference stereorandom composition. In some embodiments,provided compositions have dramatically lower complement activation. Insome embodiments, provided compositions have essentially no morecomplement activation than the negative control water.

Example 4. Example Methods for Preparing Oligonucleotides andCompositions

Oligonucleotides were diluted in distilled water to 100 μM to make stocksolution. Human Serum Albumin (Fatty acid free, Globulin free,Sigma-Aldrich A3782) was diluted with PBST (1×PBS+0.1% Tween) to 5mg/mL. Oligonucleotides were diluted 100 times into PBST or 5mg/mL-Albumin solution to make 1 μM working solution.Oligonucleotide-PBST samples provided estimates of efficiency ofoligonucleotide recovery after ultrafiltration, and were used tonormalize oligonucleotide concentrations in flow-through ofAlbumin-binding solution. The 1 μM working solutions were incubated at37° C. for half an hour. 100 μL of protein/1 μM oligonucleotide complexwas put into ultrafiltration tubes (Amicon Ultra 50 kDa cut-off,regenerated cellulose) and centrifuged at 9,000×g for 3 min. Theflow-through was collected and assayed for presence of oligonucleotides.

To detect single strand oligonucleotides, OliGreen dye was used. Thedilution buffer was TE. Each oligonucleotide had its own standard curvemade. Oligonucleotide was diluted to 0.5 μM (200×dilution from 100 μMstock), then seven 1:1 serial dilutions were made in duplicate. 20 μL ofeach diluted oligonucleotide standard was added to UV transparenthalf-area 96 well plate. All samples of ultrafiltration flow-through,including original 1 μM Oligonucleotide/PBST and 1 μMOligonucleotide/protein, were 1:1 serial diluted starting at 4 folds ofdilution. 20 μl of each sample was added along with its respectivestandard. Quant-iT OliGreen dye (Life Technologies, 07582) was diluted200 times to make working solution. 20 μl of OliGreen working solutionwas mixed with each oligonucleotide samples and incubated at roomtemperature. The plate was read using fluorescence microplate reader(excitation=480 nm; emission=520 nm).

The concentration of oligonucleotide was calculated according to its ownstandard curve. Recovery of free oligonucleotide was calculated asR_(oligo)=C_(P-FT)/C_(P-orig) (R_(oligo) is the recovery of freeoligonucleotide; CP-FT is concentration of PBST-flowthrough; C_(P-orig)is concentration of PBST original working stock). Albumin boundoligonucleotide concentration was normalized asC_(A-UB)=C_(A-FT)/R_(oligo); (C_(A-UB) is Albumin unbound oligoconcentration normalized; C_(A-FT) is measured concentration ofoligonucleotide in flow-through in Albumin treated sample). Percentageof unbound free oligonucleotide in Albumin samples was calculated asP_(A-UB)=100* C_(A-UB)/C_(A-orig) (PA-UB is percentage of unbound freeoligonucleotide of Albumin samples; C_(A-orig) is concentration ofAlbumin original working stock). % Bound=100−P_(A-UB).

Example results are presented in FIGS. 6-8. Provided compositions, forexample, WV-1092, demonstrated increased binding to albumin which canfacilitate oligonucleotide delivery.

EQUIVALENTS

Having described some illustrative embodiments of the disclosure, itshould be apparent to those skilled in the art that the foregoing ismerely illustrative and not limiting, having been presented by way ofexample only. Numerous modifications and other illustrative embodimentsare within the scope of one of ordinary skill in the art and arecontemplated as falling within the scope of the disclosure. Inparticular, although many of the examples presented herein involvespecific combinations of method acts or system elements, it should beunderstood that those acts and those elements may be combined in otherways to accomplish the same objectives. Acts, elements, and featuresdiscussed only in connection with one embodiment are not intended to beexcluded from a similar role in other embodiments. Further, for the oneor more means-plus-function limitations recited in the following claims,the means are not intended to be limited to the means disclosed hereinfor performing the recited function, but are intended to cover in scopeany means, known now or later developed, for performing the recitedfunction.

Use of ordinal terms such as “first”, “second”, “third”, etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements. Similarly, use of a), b), etc.,or i), ii), etc. does not by itself connote any priority, precedence, ororder of steps in the claims. Similarly, the use of these terms in thespecification does not by itself connote any required priority,precedence, or order.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the disclosure. The presentdisclosure is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of thedisclosure and other functionally equivalent embodiments are within thescope of the disclosure. Various modifications of the disclosure inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of thedisclosure are not necessarily encompassed by each embodiment of thedisclosure.

1-34. (canceled)
 35. A method for characterizing an oligonucleotidecomposition, the method comprising: assessing complement activationrelative to a reference composition; wherein: the oligonucleotidecomposition comprises a first plurality of oligonucleotides of aparticular oligonucleotide type defined by: 1) base sequence; 2) patternof backbone linkages; 3) pattern of backbone chiral centers; and 4)pattern of backbone phosphorus modifications. which composition ischirally controlled in that it is enriched, relative to a substantiallyracemic preparation of oligonucleotides having the same base sequence,for oligonucleotides of the particular oligonucleotide type; and thereference oligonucleotide composition is a substantially racemicpreparation of oligonucleotides that share the base sequence ofoligonucleotides of the plurality.
 36. The method of claim 35, whereinthe reference composition is a stereorandom composition ofoligonucleotides having the same base sequence and chemicalmodifications as oligonucleotides of the particular type.
 37. The methodof claim 35, wherein the oligonucleotide composition displayed reducedcomplement activation.
 38. The method of claim 35, wherein the reducedcomplement activation is observed in an assay that detects the presence,absolute level and/or relative level of C₃a or Bb.
 39. The method ofclaim 35, wherein the pattern of backbone chiral centers comprise atleast one Rp and at least one Sp.
 40. The method of claim 39, whereinthe pattern of backbone chiral centers comprise Rp(Sp)m, wherein m is 2,3, 4, 5, 6, 7 or
 8. 41. The method of claim 40, wherein the pattern ofbackbone chiral centers comprise RpSpSp.
 42. The method of claim 41,wherein the pattern of backbone chiral centers comprise (Np)t(Rp)(Sp)m,wherein t is 1-10, m is 2, 3, 4, 5, 6, 7 or 8, and each Np isindependent Rp or Sp.
 43. The method of claim 42, wherein the pattern ofbackbone chiral centers comprise SpRpSpSp.
 44. The method of claim 40,wherein oligonucleotides of the first plurality comprise one or morenatural phosphate linkages.
 45. A method for identifying anoligonucleotide composition that has reduced complement activationrelative to a reference oligonucleotide composition, the methodcomprising: providing at least one oligonucleotide compositioncomprising a first plurality of oligonucleotides; assessing complementactivation of each of the at least one oligonucleotide compositionrelative to the reference oligonucleotide composition; and identify anoligonucleotide composition that displays reduced complement activationrelative to the reference composition; wherein: the referenceoligonucleotide composition is a substantially racemic preparation ofoligonucleotides that share a common base sequence; and each of the atleast one oligonucleotide composition is independently anoligonucleotide composition comprising a first plurality ofoligonucleotides of a particular oligonucleotide type defined by: 1) thecommon base sequence; 2) pattern of backbone linkages; 3) pattern ofbackbone chiral centers; and 4) pattern of backbone phosphorusmodifications. which composition is chirally controlled in that it isenriched, relative to a substantially racemic preparation ofoligonucleotides having the same base sequence, for oligonucleotides ofthe particular oligonucleotide type.
 46. The method of claim 45, whereinthe reference composition is a stereorandom composition ofoligonucleotides having the same base sequence and chemicalmodifications as oligonucleotides of the particular type.
 47. The methodof claim 45, wherein the reduced complement activation is observed in anassay that detects the presence, absolute level and/or relative level ofC₃a or Bb.
 48. The method of claim 45, wherein the pattern of backbonechiral centers comprise at least one Rp and at least one Sp.
 49. Amethod of claim 48, wherein the pattern of backbone chiral centerscomprise Rp(Sp)m, wherein m is 2, 3, 4, 5, 6, 7 or
 8. 50. The method ofclaim 49, wherein the pattern of backbone chiral centers compriseRpSpSp.
 51. The method of claim 50, wherein the pattern of backbonechiral centers comprise (Np)t(Rp)(Sp)m, wherein t is 1-10, m is 2, 3, 4,5, 6, 7 or 8, and each Np is independent Rp or Sp.
 52. The method ofclaim 51, wherein the pattern of backbone chiral centers compriseSpRpSpSp.
 53. The method of claim 51, wherein the pattern of backbonechiral centers comprise SpSpRpSpSp.
 54. The method of claim 49, whereinoligonucleotides of the first plurality comprise one or more naturalphosphate linkages.